Human antigen binding proteins that bind β-Klotho, FGF receptors and complexes thereof

ABSTRACT

The present invention provides compositions and methods relating to or derived from antigen binding proteins activate FGF21-mediated signaling. In embodiments, the antigen binding proteins specifically bind to (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4. In some embodiments the antigen binding proteins induce FGF21-like signaling. In some embodiments, an antigen binding protein is a fully human, humanized, or chimeric antibodies, binding fragments and derivatives of such antibodies, and polypeptides that specifically bind to (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4. Other embodiments provide nucleic acids encoding such antigen binding proteins, and fragments and derivatives thereof, and polypeptides, cells comprising such polynucleotides, methods of making such antigen binding proteins, and fragments and derivatives thereof, and polypeptides, and methods of using such antigen binding proteins, fragments and derivatives thereof, and polypeptides, including methods of treating or diagnosing subjects suffering from type 2 diabetes, obesity, NASH, metabolic syndrome and related disorders or conditions.

This application claims the benefit of U.S. Provisional Application No.61/267,321 filed Dec. 7, 2009 and U.S. Provisional Application No.61/381,846 filed Sep. 10, 2010, which are incorporated by referenceherein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 24, 2010, isnamed A-1519-NP.txt and is 645,894 bytes in size.

FIELD OF THE INVENTION

The present disclosure relates to nucleic acid molecules encodingantigen binding proteins that bind to (i) β-Klotho; (ii) FGFR1c, FGFR2c,FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one ofFGFR1c, FGFR2c, FGFR3c, and FGFR4. The present disclosure also providesantigen binding proteins that bind to (i) β-Klotho; (ii) FGFR1c, FGFR2c,FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one ofFGFR1c, FGFR2c, FGFR3c, and FGFR4, including antigen binding proteinsthat induce FGF21-like signaling, as well as pharmaceutical compositionscomprising antigen binding proteins that bind to (i) β-Klotho; (ii)FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klothoand one of FGFR1c, FGFR2c, FGFR3c, and FGFR4, including antigen bindingproteins that induce FGF21-like signaling, and methods for treatingmetabolic disorders using such nucleic acids, polypeptides, orpharmaceutical compositions. Diagnostic methods using the antigenbinding proteins are also provided.

BACKGROUND

Fibroblast Growth Factor 21 (FGF21) is a secreted polypeptide thatbelongs to a subfamily of Fibroblast Growth Factors (FGFs) that includesFGF19, FGF21, and FGF23 (Itoh et al., (2004) Trend Genet. 20:563-69).FGF21 is an atypical FGF in that it is heparin independent and functionsas a hormone in the regulation of glucose, lipid, and energy metabolism.

It is highly expressed in liver and pancreas and is the only member ofthe FGF family to be primarily expressed in liver. Transgenic miceoverexpressing FGF21 exhibit metabolic phenotypes of slow growth rate,low plasma glucose and triglyceride levels, and an absence ofage-associated type 2 diabetes, islet hyperplasia, and obesity.Pharmacological administration of recombinant FGF21 protein in rodentand primate models results in normalized levels of plasma glucose,reduced triglyceride and cholesterol levels, and improved glucosetolerance and insulin sensitivity. In addition, FGF21 reduces bodyweight and body fat by increasing energy expenditure, physical activity,and metabolic rate. Experimental research provides support for thepharmacological administration of FGF21 for the treatment of type 2diabetes, obesity, dyslipidemia, and other metabolic conditions ordisorders in humans.

FGF21 is a liver derived endocrine hormone that stimulates glucoseuptake in adipocytes and lipid homeostasis through the activation of itsreceptor. Interestingly, in addition to the canonical FGF receptor, theFGF21 receptor also comprises the membrane associated β-Klotho as anessential cofactor. Activation of the FGF21 receptor leads to multipleeffects on a variety of metabolic parameters.

In mammals, FGFs mediate their action via a set of four FGF receptors,FGFR1-4, that in turn are expressed in multiple spliced variants, e.g.,FGFR1c, FGFR2c, FGFR3c and FGFR4. Each FGF receptor contains anintracellular tyrosine kinase domain that is activated upon ligandbinding, leading to downstream signaling pathways involving MAPKs(Erk1/2), RAFT, AKT1 and STATs. (Kharitonenkov et al., (2008) BioDrugs22:37-44). Several reports suggested that the “c”-reporter splicevariants of FGFR1-3 exhibit specific affinity to β-Klotho and could actas endogenous receptor for FGF21 (Kurosu et al., (2007) J. Biol. Chem.282:26687-26695); Ogawa et al., (2007) Proc. Natl. Acad. Sci. USA104:7432-7437); Kharitonenkov et al., (2008) J. Cell Physiol. 215:1-7).In the liver, which abundantly expresses both β-Klotho and FGFR4, FGF21does not induce phosphorylation of MAPK albeit the strong binding ofFGF21 to the β-Klotho-FGFR4 complex. In 3T3-L1 cells and white adiposetissue, FGFR1 is by far the most abundant receptor, and it is thereforemost likely that FGF21's main functional receptors in this tissue arethe β-Klotho/FGFR1c complexes.

The present disclosure provides a human (or humanized) antigen bindingprotein, such as a monoclonal antibody, that induces FGF21-likesignaling, e.g., an agonistic antibody that mimics the function ofFGF21. Such an antibody is a molecule with FGF21-like activity andselectivity but with added therapeutically desirable characteristicstypical for an antibody such as protein stability, lack ofimmunogenicity, ease of production and long half-life in vivo.

SUMMARY

An isolated antigen binding protein that induces FGF21-mediatedsignaling is provided.

Also provided is an isolated antigen binding protein that specificallybinds to at least one of: (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c orFGFR4; and (iii) a complex comprising β-Klotho and one of FGFR1c,FGFR2c, FGFR3c and FGFR4 wherein the antigen binding protein inducesFGF21-mediated signaling.

In one embodiment, the provided antigen binding proteins comprise anamino acid sequence selected from the group consisting of: (a) a lightchain CDR3 comprising a sequence selected from the group consisting of:(i) a light chain CDR3 sequence that differs by no more than a total ofthree amino acid additions, substitutions, and/or deletions from a CDR3sequence selected from the group consisting of the light chain CDR3sequences of L1-L18, SEQ ID NOs: 180-194; (ii) QVWDX₁X₂SDHVV (SEQ ID NO:276); (iii) QQX3GX₄X₅X₆X₇T (SEQ ID NO: 283); (iv) LQHNSYPLT (SEQ ID NO:267); (v) MQSLQTPFT (SEQ ID NO: 268); (vi) QQYNNWPPT (SEQ ID NO: 269);(vii) MQSIQLPRT (SEQ ID NO: 270); (viii) QQANDFPIT (SEQ ID NO: 271);(ix) MQALQTPCS (SEQ ID NO: 272); (b) a heavy chain CDR3 sequencecomprising a sequence selected from the group consisting of: (i) a heavychain CDR3 sequence that differs by no more than a total of four aminoacid additions, substitutions, and/or deletions from a CDR3 sequenceselected from the group consisting of the heavy chain CDR3 sequences ofH1-H18, SEQ ID NOs:145-157; (ii) X₃₄X₁₆X₁₇X₁₈GX₁₉YYYX₂₀GMDV (SEQ ID NO:322); (iii) SLIVVX₂₁VY X₂₂LDX₂₃ (SEQ ID NO: 326); (iv)IVVVPAAIQSYYYYYGMGV (SEQ ID NO: 311); (v) DPDGDYYYYGMDV (SEQ ID NO:312); (vi) TYSSGWYVWDYYGMDV (SEQ ID NO: 313); (vii) DRVLSYYAMAV (SEQ IDNO: 314); (viii) VRIAGDYY YYYGMDV (SEQ ID NO: 315); (ix)ENIVVIPAAIFAGWFDP (SEQ ID NO: 316); and (x) DRAAAGLHYYYGMDV (SEQ ID NO:317); or (c) the light chain CDR3 sequence of (a) and the heavy chainCDR3 sequence of (b); wherein, X₁ is G, S or N; X₂ is N, S or T; X₃ isC, Y or S; X₄ is G or S; X₅ is A or S; X₆ is P or F; X₇ is L or absent;X₃₄ is I, V or S; X₁₆ is L or V; X₁₇ is L, T or V; X₁₈ is L, V, G or T;X₁₉ is A, G or absent; X₂₀ is Y, C or D; X₂₁ is I or M; X₂₂ is A or V;and X₂₃ is H or Y; and wherein the antigen binding protein specificallybinds (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) acomplex comprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, andFGFR4.

In another embodiment the provided antigen binding proteins compriseeither: (a) a light chain CDR1 sequence selected from the groupconsisting of: (i) a light chain CDR1 that differs by no more than threeamino acids additions, substitutions, and/or deletions from a CDR1sequence of L1-L18, SEQ ID NOs:158-170; (ii) RASQ X₉ X₁₀X₁₁X₁₂X₁₃X₁₄LA(SEQ ID NO: 304); (iii) GGNNIGSX₁₅SVH (SEQ ID NO: 307); (iv)RSSQSLLX₂₉X₃₀NGX₃₁X₃₂X₃₃LD (SEQ ID NO: 310); (v) RASQSVNSNLA (SEQ ID NO:295); (vi) RASQDIRYDLG (SEQ ID NO: 296); (vii) RASQGISIWLA (SEQ ID NO:297); and (viii) KSSQSLLQSDGKTYLY (SEQ ID NO: 298); (b) a light chainCDR2 sequence selected from the group consisting of: (i) a light chainCDR2 that differs by no more than two amino acid additions,substitutions, and/or deletions from a CDR2 sequence of L1-L18, SEQ IDNOs:171-179; (ii) LGSX₂₇RAS (SEQ ID NO: 290); (iii) GX₈SX₂₈RAT (SEQ IDNO: 294); (iv) AASSLQS (SEQ ID NO: 284); (v) GVSTRAT (SEQ ID NO: 285);(vi) DDSDRPS (SEQ ID NO: 286); (vii) EVSNRFS (SEQ ID NO: 287); (c) aheavy chain CDR1 sequence selected from the group consisting of: (i) aheavy chain CDR1 that differs by no more than two amino acid additions,substitutions, and/or deletions from a CDR1 sequence of H1-H18, SEQ IDNOs:121-131; and (ii) NARMGVX₃₉ (SEQ ID NO: 352); (iii) X₄₀YGIH (SEQ IDNO: 355); (iv) DLSMH (SEQ ID NO: 345); (v) DAWMS (SEQ ID NO: 346); (vi)TYAMS (SEQ ID NO: 347); (vii) SYFWS (SEQ ID NO: 348); (viii) SYYWS (SEQID NO: 131); (ix) SGGYNWS (SEQ ID NO: 349); (d) a heavy chain CDR2selected from the group consisting of: (i) a heavy sequence that differsby no more than three amino acid additions, substitutions, and/ordeletions from a CDR2 sequence of H1-H18, SEQ ID NOs:132-144; (ii)HIFSNDEKSYSTSLKX₂₄ (SEQ ID NO: 333); (iii) X₂₅₁SGSGVSTX₂₆YADSVKG (SEQ IDNO: 338); (iv) VIWYDGSX₃₅KYYX₃₆DSVKG (SEQ ID NO: 341); (v)X₃₇₁YX₃₈SGSTX₄₁YNPSLKS (SEQ ID NO: 344); (vi) GFDPEDGETIYAQKFQG (SEQ IDNO: 327); (vii) RIKSKTDGGTTDYAAPVKG (SEQ ID NO: 328); (viii)RIYTSGSTNYNPSLKS (SEQ ID NO: 329); (ix) RIKSKDGGTTDYAAPVKG (SEQ ID NO:330); (x) RIKSKX₄₂DGGTTDYAAPVKG (SEQ ID NO: 483); wherein X₉ is N or S;X₁₀ is V or F; X₁₁ is D or S; X₁₂ is G or S; X₁₃ is S, N or T; X₁₄ is Sor Y; X₁₅ is E or Q; X₂₉ is Y or H; X₃₀ is Y or S; X₃₁ is F or Y; X₃₂ isT or N; X₃₃ is Y or F; X₂₇ is N or D; X₈ is A or T; X₂₈ is S or F; X₃₉is S or N; X₂₄ is S or N; X₂₅ is G or A; X₂₆ is H, Y or N; X₃₅ is D orI; X₃₆ is A or G; X₃₇ is N or R; X₃₈ is Y or T; X₄₁ is Y or N; X₄₂ is Tor absent; (e) the light chain CDR1 of (a) and the light chain CDR2 of(b); (f) the light chain CDR1 of (a) and the heavy chain CDR1 of (c);(g) the light chain CDR1 of (a) and the heavy chain CDR2 of (d); (h) thelight chain CDR1 (b) and the heavy chain CDR1 of (c); (i) the heavychain CDR1 of (c) and the heavy chain CDR2 of (d); (j) the light chainCDR2 of (b) and the heavy chain CDR2 of (d); (k) the light chain CDR1 of(a), the light chain CDR2 of (b), and the heavy chain CDR1 of (c); (l)the light chain CDR2 of (b), the heavy CDR1 of (c), and the heavy chainCDR2 of (d); (m) the light chain CDR1 of (a), the heavy chain CDR1 of(c), and the heavy chain CDR2 of (d); or (n) the light chain CDR1 of(a), the light chain CDR2 of (b), the heavy chain CDR2 of (c), and theheavy chain CDR2 of (d), wherein said antigen binding proteinspecifically binds (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4;or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c,FGFR3c, and FGFR4.

In yet another embodiment the provided antigen binding proteins compriseeither: (a) a light chain variable domain comprising; (i) a light chainCDR1 sequence selected from SEQ ID NOs:158-170; (ii) a light chain CDR2sequence selected from SEQ ID NOs:171-179; (iii) a light chain CDR3sequence selected from SEQ ID NOs:180-194; and (b) a heavy chainvariable domain comprising: (i) a heavy chain CDR1 sequence selectedfrom SEQ ID NOs:121-131; (ii) a heavy chain CDR2 sequence selected fromSEQ ID NOs:132-144; and (iii) a heavy chain CDR3 sequence selected fromSEQ ID NOs:145-157; or (c) the light chain variable domain of (a) andthe heavy chain variable domain of (b), wherein the antigen bindingprotein specifically binds (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c orFGFR4; or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c,FGFR3c, and FGFR4.

In a further embodiment the provided antigen binding proteins compriseeither: (a) a light chain variable domain sequence selected from thegroup consisting of: (i) amino acids having a sequence at least 80%identical to a light chain variable domain sequence selected fromV_(L)1-V_(L)18, SEQ ID NOs:48-65; (ii) a sequence of amino acids encodedby a polynucleotide sequence that is at least 80% identical to apolynucleotide sequence encoding the light chain variable domainsequence of V_(L)1-V_(L)18, SEQ ID NOs:48-65; (b) a heavy chain variabledomain sequence selected from the group consisting of: (i) a sequence ofamino acids that is at least 80% identical to a heavy chain variabledomain sequence of V_(H)1-V_(H)18 of SEQ ID NOs:66-84; (ii) a sequenceof amino acids encoded by a polynucleotide sequence that is at least 80%identical to a polynucleotide sequence encoding the heavy chain variabledomain sequence of V_(H)1-V_(H)18, SEQ ID NOs:66-84; or (c) the lightchain variable domain of (a) and the heavy chain variable domain of (b);wherein the antigen binding protein specifically binds (i) β-Klotho;(ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprisingβ-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4. In particularembodiments the provided antigen binding proteins comprise either: (a) alight chain variable domain sequence selected from the group consistingof: V_(L)1-V_(L)18 of SEQ ID NOs:48-65; (b) a heavy chain variabledomain sequence selected from the group consisting of: V_(H)1-V_(H)18 ofSEQ ID NOs:66-84; or (c) the light chain variable domain of (a) and theheavy chain variable domain of (b), wherein the antigen binding proteinspecifically binds (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4;or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c,FGFR3c, and FGFR4. In other particular embodiments, the provided antigenbinding proteins the light chain variable domain and a heavy chainvariable domain are selected from the group of combinations consistingof: V_(L)1V_(H)1, V_(L)2V_(H)2, V_(L)3V_(H)3, V_(L)3V_(H)4,V_(L)4V_(H)5, V_(L)5V_(H)6, V_(L)6V_(H)7, V_(L)7V_(H)8, V_(L)8V_(H)8,V_(L)9V_(H)9, V_(L)9V_(H)10, V_(L)10V_(H)11, V_(L)11V_(H)11,V_(L)12V_(H)12, V_(L)13V_(H)13, V_(L)14V_(H)14, V_(L)15V_(H)15,V_(L)16V_(H)16, V_(L)17V_(H)17, and V_(L)18V_(H)18, wherein the antigenbinding protein specifically binds (i) β-Klotho; (ii) FGFR1c, FGFR2c,FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one ofFGFR1c, FGFR2c, FGFR3c, and FGFR4. In still further embodiments theprovided antigen binding proteins further comprise: (a) the light chainconstant sequence of SEQ ID NO: 10; (b) the light chain constantsequence of SEQ ID NO:11; (c) the heavy chain constant sequence of SEQID NO: 9; or (d) the light chain constant sequence of SEQ ID NO: 10 orSEQ ID NO:11 and the heavy chain constant sequence of SEQ ID NO: 9.

The provided antigen binding proteins can take many forms and can be,for example, a human antibody, a humanized antibody, chimeric antibody,a monoclonal antibody, a polyclonal antibody, a recombinant antibody, anantigen-binding antibody fragment, a single chain antibody, a diabody, atriabody, a tetrabody, a Fab fragment, an F(fab′)₂ fragment, a domainantibody, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, or anIgG4 antibody having at least one mutation in the hinge region.

In another embodiment, the provided antigen binding proteins when boundto (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) acomplex comprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, andFGFR4: (a) bind to (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4;or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c,FGFR3c, and FGFR4, with substantially the same Kd as a referenceantibody; (b) induce FGF21-like signaling of 10% or greater than thesignaling induced by a wild-type FGF21 standard comprising the matureform of SEQ ID NO:2 as measured in an ELK-luciferase reporter assay; (c)exhibit an EC50 of 10 nM or less of FGF21-like signaling in an assayselected from the group consisting of: (i) a FGFR1c/β-Klotho-mediated invitro recombinant cell-based assay; and (ii) an in vitro human adipocytefunctional assay; (d) exhibit an EC50 of less than 10 nM of agonisticactivity on FGFR1c in the presence of β-Klotho in an in vitrorecombinant FGFR1c receptor mediated reporter assay; and (e) exhibit anEC50 of greater than 1 μM of agonistic activity on FGFR1c in the absenceof β-Klotho in an in vitro recombinant FGFR1c receptor mediated reporterassay; or (f) competes for binding with a reference antibody to (i)β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4,wherein the reference antibody comprises a combination of light chainand heavy chain variable domain sequences selected from the groupconsisting of V_(L)1V_(H)1, V_(L)2V_(H)2, V_(L)3V_(H)3, V_(L)3V_(H)4,V_(L)4V_(H)5, V_(L)5V_(H)6, V_(L)6V_(H)7, V_(L)7V_(H)8, V_(L)8V_(H)8,V_(L)9V_(H)9, V_(L)9V_(H)10, V_(L)10V_(H)11, V_(L)11V_(H)11,V_(L)12V_(H)12, V_(L)13V_(H)13, V_(L)14V_(H)14, V_(L)15V_(H)15,V_(L)16V_(H)16, V_(L)17V_(H)17, and V_(L)18V_(H)18. In other embodimentsthe provided antigen binding proteins can when bound to (i) β-Klotho;(ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprisingβ-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4: (a) lower bloodglucose in an animal model; (b) lower serum lipid levels in an animalmodel; (c) lower insulin levels in an animal model; or (d) two or moreof (a) and (b) and (c).

In specific embodiments the provided antigen binding proteins comprise:(a) a heavy chain comprising one of SEQ ID NOs:31, 32, 390-401, 404-405;(b) a light chain comprising one of SEQ ID NO:13, 14, 385-389, 402-403;or (c) a combination comprising a heavy chain of (a) and a light chainof (b).

Also provided are antigen binding proteins that are capable of bindingwild type human β-Klotho (SEQ ID NO:7) but which doesn't bind to achimeric form of β-Klotho wherein the chimeric form of β-Klothocomprises a human β-Klotho framework wherein murine β-Klotho sequencesreplace the wild type human residues at least one of (a) positions 1-80;(b) positions 303-522; (c) positions 852-1044; and (d) combinationsthereof.

In another aspect, the present disclosure provides antigen bindingproteins that are capable of binding wild type human β-Klotho (SEQ IDNO:7) at least one of (a) positions 1-80; (b) positions 303-522; (c)positions 852-1044; and (d) combinations thereof.

In still another aspect, the present disclosure provides antigen bindingproteins that are capable of competing with an antigen binding proteinof claim 8 or 13 for binding to human wild type β-Klotho residues atleast one of (a) positions 1-80; (b) positions 303-522; (c) positions852-1044; and (d) combinations thereof.

Also provided is a pharmaceutical composition comprising one or moreantigen binding proteins provided herein, in admixture with apharmaceutically acceptable carrier thereof.

In a further aspect, also provided are isolated nucleic acid moleculesthat encode the antigen binding proteins disclosed herein. In someinstances, the isolated nucleic acid molecules are operably-linked to acontrol sequence. In embodiments, such nucleic acids comprise apolynucleotide sequence encoding the light chain variable domain, theheavy chain variable domain, or both, of an antigen binding proteinprovided herein. In particular embodiments the nucleic acids comprise(a) V_(L)1-V_(L)18 (SEQ ID NOs:48-65); (b) V_(H)1-V_(H)18 (SEQ IDNOs:66-84); or (c) one or more sequences of (a) and one or moresequences of (b).

In another aspect, also provided are expression vectors and host cellstransformed or transfected with the expression vectors that comprise theaforementioned isolated nucleic acid molecules that encode the antigenbinding proteins disclosed herein.

In another aspect, also provided are methods of preparing antigenbinding proteins that specifically or selectively bind (i) β-Klotho;(ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprisingβ-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 and comprises thestep of preparing the antigen binding protein from a host cell thatsecretes the antigen binding protein.

Other embodiments provide a method of preventing or treating a conditionin a subject in need of such treatment comprising administering atherapeutically effective amount of a pharmaceutical compositionprovided herein to a subject, wherein the condition is treatable bylowering blood glucose, insulin or serum lipid levels. In embodiments,the condition is type 2 diabetes, obesity, dyslipidemia, NASH,cardiovascular disease or metabolic syndrome.

These and other aspects are described in greater detail herein. Each ofthe aspects provided can encompass various embodiments provided herein.It is therefore anticipated that each of the embodiments involving oneelement or combinations of elements can be included in each aspectdescribed, and all such combinations of the above aspects andembodiments are expressly considered. Other features, objects, andadvantages of the disclosed antigen binding proteins and associatedmethods and compositions are apparent in the detailed description thatfollows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1B is an alignment showing the sequence homology between humanFGFR1c (GenBank™ Accession No P11362; SEQ ID NO: 356) and murine FGFR1c(GenBank™ Accession No NP_(—)034336; SEQ ID NO: 357); various featuresare highlighted, including the signal peptide, transmembrane sequence,heparin binding region, and a consensus sequence (SEQ ID NO: 358) isprovided.

FIG. 2 a-2 c is an alignment showing the sequence homology between humanβ-Klotho (GenBank™ Accession No NP_(—)783864; SEQ ID NO: 359) and murineβ-Klotho (GenBank™ Accession No NP_(—)112457; SEQ ID NO: 360); variousfeatures are highlighted, including the transmembrane sequence and twoglycosyl hydrolase domains, and a consensus sequence (SEQ ID NO: 361) isprovided.

FIGS. 3A-3F are a flow cytometry profile of cells stained withFGF21-Alexa 647 that were used as an immunogen to generate antigenbinding proteins; the figure shows the expression level of an FGF21R (acomplex comprising FGFR1c and β-Klotho) and binding to FGF21.

FIG. 4 is a sequence (SEQ ID NO: 362) showing an Fc fusion protein thatwas used as an immunogen to generate antigen binding proteins; theimmunogen comprises the extracellular domain (ECD) of human FGFR1c fusedto an IgG1 Fc via a Gly₅ linker (SEQ ID NO: 379); the FGFR1c componentis in capitals, the linker is italic and underlined and the Fc is inlower case letters.

FIG. 5 is a sequence (SEQ ID NO: 363) showing an Fc fusion protein thatwas used as an immunogen to generate antigen binding proteins; theimmunogen comprises the extracellular domain (ECD) of human β-Klothofused to an IgG1 Fc via a Gly₅ linker (SEQ ID NO: 379); the β-Klothocomponent is in capitals, the linker is italic and underlined and the Fcis in lower case letters.

FIG. 6 is a SDS PAGE gel showing the level of purity achieved frompreparations of a soluble FGF21 receptor complex comprising FGFR1cECD-Fc and β-Klotho ECD-Fc, which was employed as an immunogen togenerate antigen binding proteins.

FIGS. 7A-D are a series of plots generated from an ELK-luciferasereporter assay as described herein performed on recombinant CHO clone2E10, demonstrating the ability of some of the antigen binding proteinsto induce FGF21-like signaling in recombinant CHO cells expressing aFGF21 receptor complex comprising FGFR1c and β-Klotho.

FIGS. 8A-8C are a series of plots generated from an ERK 1/2phosphorylation assay as described herein, demonstrating the ability ofsome of the antigen binding proteins to induce FGF21-like signaling inrat L6 cells. The X-axis is the concentrations of the antigen bindingproteins and the Y-axis is the percentage of phosphorylated ERK 1/2 oftotal ERK 1/2.

FIGS. 9A-9D are a series of plots generated from an ERK 1/2phosphorylation assay as described herein, demonstrating that antigenbinding protein-mediated FGF21-like signaling in L6 cells isFGFR1c/β-Klotho specific.

FIGS. 10A-10D are a series of plots generated from an ERKphosphorylation assay as described herein, demonstrating that someantigen binding proteins are able to induce FGF21-like signaling inhuman adipocyte cells.

FIGS. 11A-11C are a series of binding sensorgrams (response units vstime) demonstrating that some of the antigen binding proteins thatinduce FGF21-mediated signaling bind to human β-Klotho at two differentbut partially overlapping binding sites represented by 24H11 (Group A)and 17D8 (Group B), while antigen binding proteins that do not induceFGF21-mediated signaling (2G10, 1A2) do not bind to these sites.

FIGS. 11B-11F are a series of binding sensorgrams (response units vstime) demonstrating a third binding site on human β-Klotho that wasidentified for Group C antigen binding proteins represented by 39F7.

FIG. 11G is a table summarizing epitope binning.

FIG. 12 is a series of binding sensorgrams (response units vs time)demonstrating that some of the antigen binding proteins (12E4, 24H11,17C3, 18B11) that induce FGF21-mediated signaling interfere withβ-Klotho binding to FGF21, while other antigen binding proteins (21H2,17D8, 18G1) do not.

FIGS. 13A-13F are an alignment of the variable regions of some of theantigen binding proteins that were generated: the framework and CDRregions are identified. FIG. 13 discloses SEQ ID NOS: 364, 59, 365, 60,366, 61, 367, 62, 368, 57, 369, 55, 51-52, 56. 56, 53-54, 63-65, 370,58, 371, 50, 50, 49, 48, 372 78, 373, 66-69, 79, 374, 76, 81, 375, 70,73, 73, 71-72, 376, 83, 82, 84, 377, 80, 378, 75 and 74, respectively,in order of appearance.

FIG. 14 is a diagram graphically depicting the study design for a 68days study performed in obese cynomolgus monkeys.

FIG. 15 is a plot depicting the effects of vehicle and 16H7 on AM mealfood intake of the obese cynomolgus monkeys studied.

FIGS. 16A-16B are two plots depicting the effects of vehicle and 16H7 onfruit intake and PM food intake of the obese cynomolgus monkeys studied.

FIGS. 17A-17B are a plot depicting the effects of vehicle and 16H7 onbody weight of the obese cynomolgus monkeys studied.

FIGS. 18A-18B are a plot showing the effects of vehicle and 16H7 on bodymass index (BMI) of the obese cynomolgus monkeys studied.

FIGS. 19A-19B are a plot showing the effects of vehicle on abdominalcircumference (AC) of the obese cynomolgus monkeys studied.

FIGS. 20A-20B are a plot showing the effects of vehicle and 16H7 on skinfold thickness (SFT) of the obese cynomolgus monkeys studied.

FIGS. 21A-21D are a plot showing the effects of vehicle and 16H7 onglucose levels during glucose tolerance tests of the obese cynomolgusmonkeys studied.

FIGS. 22A-22D are a plot showing the effects of vehicle and 16H7 onplasma insulin levels during glucose tolerance tests of the obesecynomolgus monkeys studied.

FIGS. 23A-23B are a plot showing the effects of vehicle and 16H7 onfasting plasma glucose levels of the obese cynomolgus monkeys studied.

FIGS. 24A-24B are a plot showing the effects of vehicle and 16H7 onfasting plasma insulin levels of the obese cynomolgus monkeys studied.

FIGS. 25A-25B are a plot showing the effects of vehicle and 16H7 on fedplasma glucose levels of the obese cynomolgus monkeys studied.

FIGS. 26A-26B are a plot showing the effects of vehicle and 16H7 on fedplasma insulin levels of the obese cynomolgus monkeys studied.

FIGS. 27A-27B are a plot showing the effects of vehicle and 16H7 onfasting plasma triglyceride levels of the obese cynomolgus monkeysstudied.

FIGS. 28A-28B are a plot showing the effects of vehicle and 16H7 on fedplasma triglyceride levels of the obese cynomolgus monkeys studied.

FIG. 29 is a schematic depicting human-mouse β-Klotho chimeras that wereconstructed and used to studying the binding of antigen bindingproteins.

FIG. 30 is a schematic depicting the human-mouse β-Klotho chimeras thatwere constructed and also includes qualitative binding data for FGF21,16H7, 37D3 and 39F7.

FIG. 31A-C is a series of plots depicting binding data for eight of the16H7 and 22H5 variants that were constructed, as well as for 22H5 and16H7.

FIGS. 32A-C is a series of plots depicting the results of ELISA assaysthat were used to demonstrate that several of the 22H5 and 16H7 variantshave binding ability.

FIG. 33 is a bar graph comparing off-rates for several 22H5 and 17H7variants that were generated.

FIGS. 34A-34B are, two plots that depict binding curves for 39F11 whentitrated with FGF21 and for FGF21 when titrated with 39F11; the plotsdemonstrate an additive effect.

FIGS. 35A-35B are two plots that depict binding curves for 16H7 whentitrated with 39F11 and 39F11 when it is titrated with 16H7; the plotsdemonstrate an additive effect.

DETAILED DESCRIPTION

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

Generally, nomenclatures used in connection with, and techniques of,cell and tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present application are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (2001), Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates (1992), and Harlow and LaneAntibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1990), which are incorporated herein byreference. Enzymatic reactions and purification techniques are performedaccording to manufacturer's specifications, as commonly accomplished inthe art or as described herein. The terminology used in connection with,and the laboratory procedures and techniques of, analytical chemistry,synthetic organic chemistry, and medicinal and pharmaceutical chemistrydescribed herein are those well known and commonly used in the art.Standard techniques can be used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

It should be understood that the instant disclosure is not limited tothe particular methodology, protocols, and reagents, etc., describedherein and as such can vary. The terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present disclosure.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±5%, e.g., 1%, 2%, 3%, or 4%.

I. Definitions

As used herein, the terms “a” and “an” mean “one or more” unlessspecifically stated otherwise.

An “antigen binding protein” is a protein comprising a portion thatbinds to an antigen or target and, optionally, a scaffold or frameworkportion that allows the antigen binding portion to adopt a conformationthat promotes binding of the antigen binding protein to the antigen.Examples of antigen binding proteins include a human antibody, ahumanized antibody; a chimeric antibody; a recombinant antibody; asingle chain antibody; a diabody; a triabody; a tetrabody; a Fabfragment; a F(ab′)₂ fragment; an IgD antibody; an IgE antibody; an IgMantibody; an IgG1 antibody; an IgG2 antibody; an IgG3 antibody; or anIgG4 antibody, and fragments thereof. The antigen binding protein cancomprise, for example, an alternative protein scaffold or artificialscaffold with grafted CDRs or CDR derivatives. Such scaffolds include,but are not limited to, antibody-derived scaffolds comprising mutationsintroduced to, for example, stabilize the three-dimensional structure ofthe antigen binding protein as well as wholly synthetic scaffoldscomprising, for example, a biocompatible polymer. See, e.g., Korndorferet al., 2003, Proteins: Structure, Function, and Bioinformatics,53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004).In addition, peptide antibody mimetics (“PAMs”) can be used, as well asscaffolds based on antibody mimetics utilizing fibronectin components asa scaffold.

An antigen binding protein can have, for example, the structure of anaturally occurring immunoglobulin. An “immunoglobulin” is a tetramericmolecule. In a naturally occurring immunoglobulin, each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa and lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology Ch. 7 (Paul, W., ed., 2^(nd) ed. Raven Press,N.Y. (1989)) (incorporated by reference in its entirety for allpurposes). The variable regions of each light/heavy chain pair form theantibody binding site such that an intact immunoglobulin has two bindingsites.

Naturally occurring immunoglobulin chains exhibit the same generalstructure of relatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. From N-terminus to C-terminus, both light and heavy chainscomprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Theassignment of amino acids to each domain can be done in accordance withthe definitions of Kabat et al. in Sequences of Proteins ofImmunological Interest, 5^(th) Ed., US Dept. of Health and HumanServices, PHS, NIH, NIH Publication no. 91-3242, 1991. As desired, theCDRs can also be redefined according an alternative nomenclature scheme,such as that of Chothia (see Chothia & Lesk, 1987, J. Mol. Biol.196:901-917; Chothia et al., 1989, Nature 342:878-883 or Honegger &Pluckthun, 2001, J. Mol. Biol. 309:657-670).

In the context of the instant disclosure an antigen binding protein issaid to “specifically bind” or “selectively bind” its target antigenwhen the dissociation constant (K_(D)) is ≦10⁻⁸ M. The antibodyspecifically binds antigen with “high affinity” when the K_(D) is≦5×10⁻⁹ M, and with “very high affinity” when the K_(D) is ≦5×10⁻¹⁰ M.In one embodiment, the antibodies will bind to FGFR1c, β-Klotho, bothFGFR1c and β-Klotho or a complex comprising FGFR1c and β-Klotho,including human FGFR1c, human β-Klotho or both human FGFR1c and humanβ-Klotho, with a K_(D) of between about 10⁻⁷ M and 10⁻¹² M, and in yetanother embodiment the antibodies will bind with a K_(D)≦5×10⁻⁹.

An “antibody” refers to an intact immunoglobulin or to an antigenbinding portion thereof that competes with the intact antibody forspecific binding, unless otherwise specified. Antigen binding portionscan be produced by recombinant DNA techniques or by enzymatic orchemical cleavage of intact antibodies. Antigen binding portionsinclude, inter alia, Fab, Fab′, F(ab′)₂, Fv, domain antibodies (dAbs),fragments including complementarity determining regions (CDRs),single-chain antibodies (scFv), chimeric antibodies, diabodies,triabodies, tetrabodies, and polypeptides that contain at least aportion of an immunoglobulin that is sufficient to confer specificantigen binding to the polypeptide.

A Fab fragment is a monovalent fragment having the V_(L), V_(H), C_(L)and C_(H)1 domains; a F(ab′)₂ fragment is a bivalent fragment having twoFab fragments linked by a disulfide bridge at the hinge region; a Fdfragment has the V_(H) and C_(H)1 domains; an Fv fragment has the V_(L)and V_(H) domains of a single arm of an antibody; and a dAb fragment hasa V_(H) domain, a V_(L) domain, or an antigen-binding fragment of aV_(H) or V_(L) domain (U.S. Pat. Nos. 6,846,634, 6,696,245, U.S.application Pub. Ser. Nos. 05/0202512, 04/0202995, 04/0038291,04/0009507, 03/0039958, Ward et al., Nature 341:544-546 (1989)).

A single-chain antibody (scFv) is an antibody in which a V_(L) and aV_(H) region are joined via a linker (e.g., a synthetic sequence ofamino acid residues) to form a continuous protein chain wherein thelinker is long enough to allow the protein chain to fold back on itselfand form a monovalent antigen binding site (see, e.g., Bird et al.,Science 242:423-26 (1988) and Huston et al., 1988, Proc. Natl. Acad.Sci. USA 85:5879-83 (1988)). Diabodies are bivalent antibodiescomprising two polypeptide chains, wherein each polypeptide chaincomprises V_(H) and V_(L) domains joined by a linker that is too shortto allow for pairing between two domains on the same chain, thusallowing each domain to pair with a complementary domain on anotherpolypeptide chain (see, e.g., Holliger et al., 1993, Proc. Natl. Acad.Sci. USA 90:6444-48 (1993), and Poljak et al., Structure 2:1121-23(1994)). If the two polypeptide chains of a diabody are identical, thena diabody resulting from their pairing will have two identical antigenbinding sites. Polypeptide chains having different sequences can be usedto make a diabody with two different antigen binding sites. Similarly,tribodies and tetrabodies are antibodies comprising three and fourpolypeptide chains, respectively, and forming three and four antigenbinding sites, respectively, which can be the same or different.

Complementarity determining regions (CDRs) and framework regions (FR) ofa given antibody can be identified using the system described by Kabatet al. in Sequences of Proteins of Immunological Interest, 5th Ed., USDept. of Health and Human Services, PHS, NIH, NIH Publication no.91-3242, 1991 As desired, the CDRs can also be redefined according analternative nomenclature scheme, such as that of Chothia (see Chothia &Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature342:878-883 or Honegger & Pluckthun, 2001, J. Mol. Biol. 309:657-670.One or more CDRs can be incorporated into a molecule either covalentlyor noncovalently to make it an antigen binding protein. An antigenbinding protein can incorporate the CDR(s) as part of a largerpolypeptide chain, can covalently link the CDR(s) to another polypeptidechain, or can incorporate the CDR(s) noncovalently. The CDRs permit theantigen binding protein to specifically bind to a particular antigen ofinterest.

An antigen binding protein can have one or more binding sites. If thereis more than one binding site, the binding sites can be identical to oneanother or can be different. For example, a naturally occurring humanimmunoglobulin typically has two identical binding sites, while a“bispecific” or “bifunctional” antibody has two different binding sites.

The term “human antibody” includes all antibodies that have one or morevariable and constant regions derived from human immunoglobulinsequences. In one embodiment, all of the variable and constant domainsare derived from human immunoglobulin sequences (a fully humanantibody). These antibodies can be prepared in a variety of ways,examples of which are described below, including through theimmunization with an antigen of interest of a mouse that is geneticallymodified to express antibodies derived from human heavy and/or lightchain-encoding genes, such as a mouse derived from a Xenomouse®,UltiMab™, or Velocimmune® system. Phage-based approaches can also beemployed.

A humanized antibody has a sequence that differs from the sequence of anantibody derived from a non-human species by one or more amino acidsubstitutions, deletions, and/or additions, such that the humanizedantibody is less likely to induce an immune response, and/or induces aless severe immune response, as compared to the non-human speciesantibody, when it is administered to a human subject. In one embodiment,certain amino acids in the framework and constant domains of the heavyand/or light chains of the non-human species antibody are mutated toproduce the humanized antibody. In another embodiment, the constantdomain(s) from a human antibody are fused to the variable domain(s) of anon-human species. In another embodiment, one or more amino acidresidues in one or more CDR sequences of a non-human antibody arechanged to reduce the likely immunogenicity of the non-human antibodywhen it is administered to a human subject, wherein the changed aminoacid residues either are not critical for immunospecific binding of theantibody to its antigen, or the changes to the amino acid sequence thatare made are conservative changes, such that the binding of thehumanized antibody to the antigen is not significantly worse than thebinding of the non-human antibody to the antigen. Examples of how tomake humanized antibodies can be found in U.S. Pat. Nos. 6,054,297,5,886,152 and 5,877,293.

The term “chimeric antibody” refers to an antibody that contains one ormore regions from one antibody and one or more regions from one or moreother antibodies. In one embodiment, one or more of the CDRs are derivedfrom a human antibody that binds (i) β-Klotho; (ii) FGFR1c, FGFR2c,FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one ofFGFR1c, FGFR2c, FGFR3c, and FGFR4. In another embodiment, all of theCDRs are derived from a human antibody that binds (i) β-Klotho; (ii)FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klothoand one of FGFR1c, FGFR2c, FGFR3c, and FGFR4. In another embodiment, theCDRs from more than one human antibody that binds (i) β-Klotho; (ii)FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klothoand one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 are mixed and matched in achimeric antibody. For instance, a chimeric antibody can comprise a CDR1from the light chain of a first human antibody that binds (i) β-Klotho;(ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprisingβ-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4, a CDR2 and a CDR3from the light chain of a second human antibody that binds (i) β-Klotho;(ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprisingβ-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4, and the CDRs fromthe heavy chain from a third antibody that binds (i) β-Klotho; (ii)FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klothoand one of FGFR1c, FGFR2c, FGFR3c, and FGFR4. Further, the frameworkregions can be derived from one of the same antibodies that bind (i)β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4, fromone or more different antibodies, such as a human antibody, or from ahumanized antibody. In one example of a chimeric antibody, a portion ofthe heavy and/or light chain is identical with, homologous to, orderived from an antibody from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is/are identical with, homologous to, or derived from anantibody or antibodies from another species or belonging to anotherantibody class or subclass. Also included are fragments of suchantibodies that exhibit the desired biological activity (e.g., theability to specifically bind (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3cor FGFR4; or (iii) a complex comprising β-Klotho and one of FGFR1c,FGFR2c, FGFR3c, and FGFR4).

The term “light chain” includes a full-length light chain and fragmentsthereof having sufficient variable region sequence to confer bindingspecificity. A full-length light chain includes a variable regiondomain, V_(L), and a constant region domain, C_(L). The variable regiondomain of the light chain is at the amino-terminus of the polypeptide.Light chains include kappa (“κ”) chains and lambda (“λ”) chains.

The term “heavy chain” includes a full-length heavy chain and fragmentsthereof having sufficient variable region sequence to confer bindingspecificity. A full-length heavy chain includes a variable regiondomain, V_(H), and three constant region domains, C_(H)1, C_(H)2, andC_(H)3. The V_(H) domain is at the amino-terminus of the polypeptide,and the C_(H) domains are at the carboxyl-terminus, with the C_(H)3being closest to the carboxy-terminus of the polypeptide. Heavy chainscan be of any isotype, including IgG (including IgG1, IgG2, IgG3 andIgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.

The term “immunologically functional fragment” (or simply “fragment”) ofan antigen binding protein, e.g., an antibody or immunoglobulin chain(heavy or light chain), as used herein, is an antigen binding proteincomprising a portion (regardless of how that portion is obtained orsynthesized) of an antibody that lacks at least some of the amino acidspresent in a full-length chain but which is capable of specificallybinding to an antigen. Such fragments are biologically active in thatthey bind specifically to the target antigen and can compete with otherantigen binding proteins, including intact antibodies, for specificbinding to a given epitope. In one aspect, such a fragment will retainat least one CDR present in the full-length light or heavy chain, and insome embodiments will comprise a single heavy chain and/or light chainor portion thereof. These biologically active fragments can be producedby recombinant DNA techniques, or can be produced by enzymatic orchemical cleavage of antigen binding proteins, including intactantibodies. Immunologically functional immunoglobulin fragments include,but are not limited to, Fab, Fab′, F(ab′)₂, Fv, domain antibodies andsingle-chain antibodies, and can be derived from any mammalian source,including but not limited to human, mouse, rat, camelid or rabbit. It iscontemplated further that a functional portion of the antigen bindingproteins disclosed herein, for example, one or more CDRs, could becovalently bound to a second protein or to a small molecule to create atherapeutic agent directed to a particular target in the body,possessing bifunctional therapeutic properties, or having a prolongedserum half-life.

An “Fc” region contains two heavy chain fragments comprising the C_(H)2and C_(H)3 domains of an antibody. The two heavy chain fragments areheld together by two or more disulfide bonds and by hydrophobicinteractions of the C_(H)3 domains.

An “Fab′ fragment” contains one light chain and a portion of one heavychain that contains the V_(H) domain and the C_(H)1 domain and also theregion between the C_(H)1 and C_(H)2 domains, such that an interchaindisulfide bond can be formed between the two heavy chains of two Fab′fragments to form an F(ab′)₂ molecule.

An “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H)1 andC_(H)2 domains, such that an interchain disulfide bond is formed betweenthe two heavy chains. A F(ab′)₂ fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the twoheavy chains.

The “Fv region” comprises the variable regions from both the heavy andlight chains, but lacks the constant regions.

A “domain antibody” is an immunologically functional immunoglobulinfragment containing only the variable region of a heavy chain or thevariable region of a light chain. In some instances, two or more V_(H)regions are covalently joined with a peptide linker to create a bivalentdomain antibody. The two V_(H) regions of a bivalent domain antibody cantarget the same or different antigens.

A “hemibody” is an immunologically functional immunoglobulin constructcomprising a complete heavy chain, a complete light chain and a secondheavy chain Fc region paired with the Fc region of the complete heavychain. A linker can, but need not, be employed to join the heavy chainFc region and the second heavy chain Fc region. In particularembodiments a hemibody is a monovalent form of an antigen bindingprotein disclosed herein. In other embodiments, pairs of chargedresidues can be employed to associate one Fc region with the second Fcregion. The second heavy chain Fc region can comprise, for example, SEQID NO:441 and can be joined to the light chain via a linker (e.g., SEQID NO:440) An exemplary hemibody heavy chain comprises the sequence SEQID NO:453.

A “bivalent antigen binding protein” or “bivalent antibody” comprisestwo antigen binding sites. In some instances, the two binding sites havethe same antigen specificities. Bivalent antigen binding proteins andbivalent antibodies can be bispecific, as described herein.

A multispecific antigen binding protein” or “multispecific antibody” isone that targets more than one antigen or epitope.

A “bispecific,” “dual-specific” or “bifunctional” antigen bindingprotein or antibody is a hybrid antigen binding protein or antibody,respectively, having two different antigen binding sites. Bispecificantigen binding proteins and antibodies are a species of multispecificantigen binding protein or multispecific antibody and can be produced bya variety of methods including, but not limited to, fusion of hybridomasor linking of Fab′ fragments. See, e.g., Songsivilai and Lachmann, 1990,Clin. Exp. Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol.148:1547-1553. The two binding sites of a bispecific antigen bindingprotein or antibody will bind to two different epitopes, which canreside on the same or different protein targets.

The terms “FGF21-like signaling” and “induces FGF21-like signaling,”when applied to an antigen binding protein of the present disclosure,means that the antigen binding protein mimics, or modulates, an in vivobiological effect induced by the binding of (i) β-Klotho; (ii) FGFR1c,FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and oneof FGFR1c, FGFR2c, FGFR3c, and FGFR4 and induces a biological responsethat otherwise would result from FGF21 binding to (i) β-Klotho; (ii)FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klothoand one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 in vivo. In assessing thebinding and specificity of an antigen binding protein, e.g., an antibodyor immunologically functional fragment thereof, an antibody or fragmentis deemed to induce a biological response when the response is equal toor greater than 5%, and preferably equal to or greater than 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90% or 95%, of the activity of a wild type FGF21 standard comprising themature form of SEQ ID NO:2 (i.e., the mature form of the human FGF21sequence) and has the following properties: exhibiting an efficacy levelof equal to or more than 5% of an FGF21 standard, with an EC50 of equalto or less than 100 nM, e.g., 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM,30 nM, 20 nM or 10 nM in (1) the recombinant FGF21 receptor mediatedluciferase-reporter cell assay of Example 5; (2) ERK-phosphorylation inthe recombinant FGF21 receptor mediated cell assay of Example 5; and (3)ERK-phosphorylation in human adipocytes as described in Example 7. The“potency” of an antigen binding protein is defined as exhibiting an EC50of equal to or less than 100 nM, e.g., 90 nM, 80 nM, 70 nM, 60 nM, 50nM, 40 nM, 30 nM, 20 nM, 10 nM and preferably less than 10 nM of theantigen binding protein in the following assays: (1) the recombinantFGF21 receptor mediated luciferase-reporter cell assay of Example 5; (2)the ERK-phosphorylation in the recombinant FGF21 receptor mediated cellassay of Example 5; and (3) ERK-phosphorylation in human adipocytes asdescribed in Example 7.

It is noted that not all of the antigen binding proteins of the presentdisclosure induce FGF21-mediated signaling, nor is this propertydesirable in all circumstances. Nevertheless, antigen binding proteinsthat do not induce FGF21-mediated signaling form aspects of the presentdisclosure and may be useful as diagnostic reagents or otherapplications.

As used herein, the term “FGF21R” means a multimeric receptor complexthat FGF21 is known or suspected to form in vivo. In variousembodiments, FGF21R comprises (i) an FGFR, e.g., FGFR1c, FGFR2c, FGFR3cor FGFR4, and (ii) β-Klotho.

The term “polynucleotide” or “nucleic acid” includes bothsingle-stranded and double-stranded nucleotide polymers. The nucleotidescomprising the polynucleotide can be ribonucleotides ordeoxyribonucleotides or a modified form of either type of nucleotide.Said modifications include base modifications such as bromouridine andinosine derivatives, ribose modifications such as 2′,3′-dideoxyribose,and internucleotide linkage modifications such as phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phoshoraniladate and phosphoroamidate.

The term “oligonucleotide” means a polynucleotide comprising 200 orfewer nucleotides. In some embodiments, oligonucleotides are 10 to 60bases in length. In other embodiments, oligonucleotides are 12, 13, 14,15, 16, 17, 18, 19, or 20 to 40 nucleotides in length. Oligonucleotidescan be single stranded or double stranded, e.g., for use in theconstruction of a mutant gene. Oligonucleotides can be sense orantisense oligonucleotides. An oligonucleotide can include a label,including a radiolabel, a fluorescent label, a hapten or an antigeniclabel, for detection assays. Oligonucleotides can be used, for example,as PCR primers, cloning primers or hybridization probes.

An “isolated nucleic acid molecule” means a DNA or RNA of genomic, mRNA,cDNA, or synthetic origin or some combination thereof which is notassociated with all or a portion of a polynucleotide in which theisolated polynucleotide is found in nature, or is linked to apolynucleotide to which it is not linked in nature. For purposes of thisdisclosure, it is understood that “a nucleic acid molecule comprising” aparticular nucleotide sequence does not encompass intact chromosomes.Isolated nucleic acid molecules “comprising” specified nucleic acidsequences can include, in addition to the specified sequences, codingsequences for up to ten or even up to twenty other proteins or portionsthereof, or can include operably linked regulatory sequences thatcontrol expression of the coding region of the recited nucleic acidsequences, and/or can include vector sequences.

Unless specified otherwise, the left-hand end of any single-strandedpolynucleotide sequence discussed herein is the 5′ end; the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA transcriptthat are 5′ to the 5′ end of the RNA transcript are referred to as“upstream sequences;” sequence regions on the DNA strand having the samesequence as the RNA transcript that are 3′ to the 3′ end of the RNAtranscript are referred to as “downstream sequences.”

The term “control sequence” refers to a polynucleotide sequence that canaffect the expression and processing of coding sequences to which it isligated. The nature of such control sequences can depend upon the hostorganism. In particular embodiments, control sequences for prokaryotescan include a promoter, a ribosomal binding site, and a transcriptiontermination sequence. For example, control sequences for eukaryotes caninclude promoters comprising one or a plurality of recognition sites fortranscription factors, transcription enhancer sequences, andtranscription termination sequence. “Control sequences” can includeleader sequences and/or fusion partner sequences.

The term “vector” means any molecule or entity (e.g., nucleic acid,plasmid, bacteriophage or virus) used to transfer protein codinginformation into a host cell.

The term “expression vector” or “expression construct” refers to avector that is suitable for transformation of a host cell and containsnucleic acid sequences that direct and/or control (in conjunction withthe host cell) expression of one or more heterologous coding regionsoperatively linked thereto. An expression construct can include, but isnot limited to, sequences that affect or control transcription,translation, and, if introns are present, affect RNA splicing of acoding region operably linked thereto.

As used herein, “operably linked” means that the components to which theterm is applied are in a relationship that allows them to carry outtheir inherent functions under suitable conditions. For example, acontrol sequence in a vector that is “operably linked” to a proteincoding sequence is ligated thereto so that expression of the proteincoding sequence is achieved under conditions compatible with thetranscriptional activity of the control sequences.

The term “host cell” means a cell that has been transformed, or iscapable of being transformed, with a nucleic acid sequence and therebyexpresses a gene of interest. The term includes the progeny of theparent cell, whether or not the progeny is identical in morphology or ingenetic make-up to the original parent cell, so long as the gene ofinterest is present.

The term “transduction” means the transfer of genes from one bacteriumto another, usually by bacteriophage. “Transduction” also refers to theacquisition and transfer of eukaryotic cellular sequences byreplication-defective retroviruses.

The term “transfection” means the uptake of foreign or exogenous DNA bya cell, and a cell has been “transfected” when the exogenous DNA hasbeen introduced inside the cell membrane. A number of transfectiontechniques are well known in the art and are disclosed herein. See,e.g., Graham et al., (1973) Virology 52:456; Sambrook et al., (2001)Molecular Cloning: A Laboratory Manual, supra; Davis et al., (1986)Basic Methods in Molecular Biology, Elsevier; Chu et al., (1981) Gene13:197. Such techniques can be used to introduce one or more exogenousDNA moieties into suitable host cells.

The term “transformation” refers to a change in a cell's geneticcharacteristics, and a cell has been transformed when it has beenmodified to contain new DNA or RNA. For example, a cell is transformedwhere it is genetically modified from its native state by introducingnew genetic material via transfection, transduction, or othertechniques. Following transfection or transduction, the transforming DNAcan recombine with that of the cell by physically integrating into achromosome of the cell, or can be maintained transiently as an episomalelement without being replicated, or can replicate independently as aplasmid. A cell is considered to have been “stably transformed” when thetransforming DNA is replicated with the division of the cell.

The terms “polypeptide” or “protein” are used interchangeably herein torefer to a polymer of amino acid residues. The terms also apply to aminoacid polymers in which one or more amino acid residues is an analog ormimetic of a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers. The terms can also encompassamino acid polymers that have been modified, e.g., by the addition ofcarbohydrate residues to form glycoproteins, or phosphorylated.Polypeptides and proteins can be produced by a naturally-occurring andnon-recombinant cell, or polypeptides and proteins can be produced by agenetically-engineered or recombinant cell. Polypeptides and proteinscan comprise molecules having the amino acid sequence of a nativeprotein, or molecules having deletions from, additions to, and/orsubstitutions of one or more amino acids of the native sequence. Theterms “polypeptide” and “protein” encompass antigen binding proteinsthat specifically or selectively bind (i) β-Klotho; (ii) FGFR1c, FGFR2c,FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one ofFGFR1c, FGFR2c, FGFR3c, and FGFR4, or sequences that have deletionsfrom, additions to, and/or substitutions of one or more amino acids ofan antigen binding protein that specifically or selectively binds (i)β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4. Theterm “polypeptide fragment” refers to a polypeptide that has anamino-terminal deletion, a carboxyl-terminal deletion, and/or aninternal deletion as compared with the full-length protein. Suchfragments can also contain modified amino acids as compared with thefull-length protein. In certain embodiments, fragments are about five to500 amino acids long. For example, fragments can be at least 5, 6, 8,10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 aminoacids long. Useful polypeptide fragments include immunologicallyfunctional fragments of antibodies, including binding domains. In thecase of an antigen binding protein that binds to (i) β-Klotho; (ii)FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klothoand one of FGFR1c, FGFR2c, FGFR3c, and FGFR4, useful fragments includebut are not limited to a CDR region, a variable domain of a heavy orlight chain, a portion of an antibody chain or just its variable regionincluding two CDRs, and the like.

The term “isolated protein” referred means that a subject protein (1) isfree of at least some other proteins with which it would normally befound, (2) is essentially free of other proteins from the same source,e.g., from the same species, (3) is expressed by a cell from a differentspecies, (4) has been separated from at least about 50 percent ofpolynucleotides, lipids, carbohydrates, or other materials with which itis associated in nature, (5) is operably associated (by covalent ornoncovalent interaction) with a polypeptide with which it is notassociated in nature, or (6) does not occur in nature. Typically, an“isolated protein” constitutes at least about 5%, at least about 10%, atleast about 25%, or at least about 50% of a given sample. Genomic DNA,cDNA, mRNA or other RNA, of synthetic origin, or any combination thereofcan encode such an isolated protein. Preferably, the isolated protein issubstantially free from proteins or polypeptides or other contaminantsthat are found in its natural environment that would interfere with itstherapeutic, diagnostic, prophylactic, research or other use.

A “variant” of a polypeptide (e.g., an antigen binding protein, or anantibody) comprises an amino acid sequence wherein one or more aminoacid residues are inserted into, deleted from and/or substituted intothe amino acid sequence relative to another polypeptide sequence.Variants include fusion proteins.

A “derivative” of a polypeptide is a polypeptide (e.g., an antigenbinding protein, or an antibody) that has been chemically modified insome manner distinct from insertion, deletion, or substitution variants,e.g., by conjugation to another chemical moiety.

The term “naturally occurring” as used throughout the specification inconnection with biological materials such as polypeptides, nucleicacids, host cells, and the like, refers to materials which are found innature.

“Antigen binding region” means a protein, or a portion of a protein,that specifically binds a specified antigen, e.g., FGFR1c, β-Klotho orboth FGFR1c and β-Klotho. For example, that portion of an antigenbinding protein that contains the amino acid residues that interact withan antigen and confer on the antigen binding protein its specificity andaffinity for the antigen is referred to as “antigen binding region.” Anantigen binding region typically includes one or more “complementarybinding regions” (“CDRs”). Certain antigen binding regions also includeone or more “framework” regions. A “CDR” is an amino acid sequence thatcontributes to antigen binding specificity and affinity. “Framework”regions can aid in maintaining the proper conformation of the CDRs topromote binding between the antigen binding region and an antigen.

In certain aspects, recombinant antigen binding proteins that bind (i)β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4, areprovided. In this context, a “recombinant protein” is a protein madeusing recombinant techniques, i.e., through the expression of arecombinant nucleic acid as described herein. Methods and techniques forthe production of recombinant proteins are well known in the art.

The term “compete” when used in the context of antigen binding proteins(e.g., neutralizing antigen binding proteins, neutralizing antibodies,agonistic antigen binding proteins, agonistic antibodies and bindingproteins that bind to (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c orFGFR4; or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c,FGFR3c, and FGFR4) that compete for the same epitope or binding site ona target means competition between antigen binding proteins asdetermined by an assay in which the antigen binding protein (e.g.,antibody or immunologically functional fragment thereof) under studyprevents or inhibits the specific binding of a reference molecule (e.g.,a reference ligand, or reference antigen binding protein, such as areference antibody) to a common antigen (e.g., FGFR1c, FGFR2c, FGFR3c,FGFR4, β-Klotho or a fragment thereof). Numerous types of competitivebinding assays can be used to determine if a test molecule competes witha reference molecule for binding. Examples of assays that can beemployed include solid phase direct or indirect radioimmunoassay (RIA),solid phase direct or indirect enzyme immunoassay (EIA), sandwichcompetition assay (see, e.g., Stahli et al., (1983) Methods inEnzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g.,Kirkland et al., (1986) J. Immunol. 137:3614-3619) solid phase directlabeled assay, solid phase direct labeled sandwich assay (see, e.g.,Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold SpringHarbor Press); solid phase direct label RIA using 1-125 label (see,e.g., Morel et al., (1988) Molec. Immunol. 25:7-15); solid phase directbiotin-avidin EIA (see, e.g., Cheung, et al., (1990) Virology176:546-552); and direct labeled RIA (Moldenhauer et al., (1990) Scand.J. Immunol. 32:77-82). Typically, such an assay involves the use of apurified antigen bound to a solid surface or cells bearing either of anunlabelled test antigen binding protein or a labeled reference antigenbinding protein. Competitive inhibition is measured by determining theamount of label bound to the solid surface or cells in the presence ofthe test antigen binding protein. Usually the test antigen bindingprotein is present in excess. Antigen binding proteins identified bycompetition assay (competing antigen binding proteins) include antigenbinding proteins binding to the same epitope as the reference antigenbinding proteins and antigen binding proteins binding to an adjacentepitope sufficiently proximal to the epitope bound by the referenceantigen binding protein for steric hindrance to occur. Additionaldetails regarding methods for determining competitive binding areprovided in the examples herein. Usually, when a competing antigenbinding protein is present in excess, it will inhibit specific bindingof a reference antigen binding protein to a common antigen by at least40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some instance, binding isinhibited by at least 80%, 85%, 90%, 95%, or 97% or more.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as an antigenbinding protein (including, e.g., an antibody or immunologicalfunctional fragment thereof), and may also be capable of being used inan animal to produce antibodies capable of binding to that antigen. Anantigen can possess one or more epitopes that are capable of interactingwith different antigen binding proteins, e.g., antibodies.

The term “epitope” means the amino acids of a target molecule that arecontacted by an antigen binding protein (for example, an antibody) whenthe antigen binding protein is bound to the target molecule. The termincludes any subset of the complete list of amino acids of the targetmolecule that are contacted when an antigen binding protein, such as anantibody, is bound to the target molecule. An epitope can be contiguousor non-contiguous (e.g., (i) in a single-chain polypeptide, amino acidresidues that are not contiguous to one another in the polypeptidesequence but that within in context of the target molecule are bound bythe antigen binding protein, or (ii) in a multimeric receptor comprisingtwo or more individual components, e.g., (i) FGFR1c, FGFR2c, FGFR3c orFGFR4, and (ii) β-Klotho, amino acid residues that are present on one ormore of the individual components, but which are still bound by theantigen binding protein). In certain embodiments, epitopes can bemimetic in that they comprise a three dimensional structure that issimilar to an antigenic epitope used to generate the antigen bindingprotein, yet comprise none or only some of the amino acid residues foundin that epitope used to generate the antigen binding protein. Mostoften, epitopes reside on proteins, but in some instances can reside onother kinds of molecules, such as nucleic acids. Epitope determinantscan include chemically active surface groupings of molecules such asamino acids, sugar side chains, phosphoryl or sulfonyl groups, and canhave specific three dimensional structural characteristics, and/orspecific charge characteristics. Generally, antigen binding proteinsspecific for a particular target molecule will preferentially recognizean epitope on the target molecule in a complex mixture of proteinsand/or macromolecules.

The term “identity” refers to a relationship between the sequences oftwo or more polypeptide molecules or two or more nucleic acid molecules,as determined by aligning and comparing the sequences. “Percentidentity” means the percent of identical residues between the aminoacids or nucleotides in the compared molecules and is calculated basedon the size of the smallest of the molecules being compared. For thesecalculations, gaps in alignments (if any) must be addressed by aparticular mathematical model or computer program (i.e., an“algorithm”). Methods that can be used to calculate the identity of thealigned nucleic acids or polypeptides include those described inComputational Molecular Biology, (Lesk, A. M., ed.), (1988) New York:Oxford University Press; Biocomputing Informatics and Genome Projects,(Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysisof Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.),1994, New Jersey: Humana Press; von Heinje, G., (1987) Sequence Analysisin Molecular Biology, New York: Academic Press; Sequence AnalysisPrimer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M.Stockton Press; and Carillo et al., (1988) SIAM J. Applied Math.48:1073.

In calculating percent identity, the sequences being compared arealigned in a way that gives the largest match between the sequences. Thecomputer program used to determine percent identity is the GCG programpackage, which includes GAP (Devereux et al., (1984) Nucl. Acid Res.12:387; Genetics Computer Group, University of Wisconsin, Madison,Wis.). The computer algorithm GAP is used to align the two polypeptidesor polynucleotides for which the percent sequence identity is to bedetermined. The sequences are aligned for optimal matching of theirrespective amino acid or nucleotide (the “matched span”, as determinedby the algorithm). A gap opening penalty (which is calculated as 3× theaverage diagonal, wherein the “average diagonal” is the average of thediagonal of the comparison matrix being used; the “diagonal” is thescore or number assigned to each perfect amino acid match by theparticular comparison matrix) and a gap extension penalty (which isusually 1/10 times the gap opening penalty), as well as a comparisonmatrix such as PAM 250 or BLOSUM 62 are used in conjunction with thealgorithm. In certain embodiments, a standard comparison matrix (see,Dayhoff et al., (1978) Atlas of Protein Sequence and Structure 5:345-352for the PAM 250 comparison matrix; Henikoff et al., (1992) Proc. Natl.Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) isalso used by the algorithm.

Recommended parameters for determining percent identity for polypeptidesor nucleotide sequences using the GAP program are the following:

Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453;

Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra;

Gap Penalty: 12 (but with no penalty for end gaps)

Gap Length Penalty: 4

Threshold of Similarity: 0

Certain alignment schemes for aligning two amino acid sequences canresult in matching of only a short region of the two sequences, and thissmall aligned region can have very high sequence identity even thoughthere is no significant relationship between the two full-lengthsequences. Accordingly, the selected alignment method (e.g., the GAPprogram) can be adjusted if so desired to result in an alignment thatspans at least 50 contiguous amino acids of the target polypeptide.

As used herein, “substantially pure” means that the described species ofmolecule is the predominant species present, that is, on a molar basisit is more abundant than any other individual species in the samemixture. In certain embodiments, a substantially pure molecule is acomposition wherein the object species comprises at least 50% (on amolar basis) of all macromolecular species present. In otherembodiments, a substantially pure composition will comprise at least80%, 85%, 90%, 95%, or 99% of all macromolecular species present in thecomposition. In other embodiments, the object species is purified toessential homogeneity wherein contaminating species cannot be detectedin the composition by conventional detection methods and thus thecomposition consists of a single detectable macromolecular species.

The terms “treat” and “treating” refer to any indicia of success in thetreatment or amelioration of an injury, pathology or condition,including any objective or subjective parameter such as abatement;remission; diminishing of symptoms or making the injury, pathology orcondition more tolerable to the patient; slowing in the rate ofdegeneration or decline; making the final point of degeneration lessdebilitating; improving a patient's physical or mental well-being. Thetreatment or amelioration of symptoms can be based on objective orsubjective parameters; including the results of a physical examination,neuropsychiatric exams, and/or a psychiatric evaluation. For example,certain methods presented herein can be employed to treat Type 2diabetes, obesity and/or dyslipidemia, either prophylactically or as anacute treatment, to decrease plasma glucose levels, to decreasecirculating triglyceride levels, to decrease circulating cholesterollevels and/or ameliorate a symptom associated with type 2 diabetes,obesity and dyslipidemia.

An “effective amount” is generally an amount sufficient to reduce theseverity and/or frequency of symptoms, eliminate the symptoms and/orunderlying cause, prevent the occurrence of symptoms and/or theirunderlying cause, and/or improve or remediate the damage that resultsfrom or is associated with diabetes, obesity and dyslipidemia. In someembodiments, the effective amount is a therapeutically effective amountor a prophylactically effective amount. A “therapeutically effectiveamount” is an amount sufficient to remedy a disease state (e.g.,diabetes, obesity or dyslipidemia) or symptoms, particularly a state orsymptoms associated with the disease state, or otherwise prevent,hinder, retard or reverse the progression of the disease state or anyother undesirable symptom associated with the disease in any waywhatsoever. A “prophylactically effective amount” is an amount of apharmaceutical composition that, when administered to a subject, willhave the intended prophylactic effect, e.g., preventing or delaying theonset (or reoccurrence) of diabetes, obesity or dyslipidemia, orreducing the likelihood of the onset (or reoccurrence) of diabetes,obesity or dyslipidemia or associated symptoms. The full therapeutic orprophylactic effect does not necessarily occur by administration of onedose, and can occur only after administration of a series of doses.Thus, a therapeutically or prophylactically effective amount can beadministered in one or more administrations.

“Amino acid” takes its normal meaning in the art. The twentynaturally-occurring amino acids and their abbreviations followconventional usage. See, Immunology-A Synthesis, 2^(nd) Edition, (E. S.Golub and D. R. Green, eds.), Sinauer Associates: Sunderland, Mass.(1991), incorporated herein by reference for any purpose. Stereoisomers(e.g., D-amino acids) of the twenty conventional amino acids, unnaturalor non-naturally occurring amino acids such as α-,α-disubstituted aminoacids, N-alkyl amino acids, and other unconventional amino acids canalso be suitable components for polypeptides and are included in thephrase “amino acid.” Examples of non-naturally amino acids (which can besubstituted for any naturally-occurring amino acid found in any sequencedisclosed herein, as desired) include: 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, σ-N-methylarginine, and other similar amino acids andimino acids (e.g., 4-hydroxyproline). In the polypeptide notation usedherein, the left-hand direction is the amino terminal direction and theright-hand direction is the carboxyl-terminal direction, in accordancewith standard usage and convention. A non-limiting lists of examples ofnon-naturally occurring amino acids that can be inserted into an antigenbinding protein sequence or substituted for a wild-type residue in anantigen binding sequence include β-amino acids, homoamino acids, cyclicamino acids and amino acids with derivatized side chains. Examplesinclude (in the L-form or D-form; abbreviated as in parentheses):citrulline (Cit), homocitrulline (hCit), Nα-methylcitrulline (NMeCit),Nα-methylhomocitrulline (Nα-MeHoCit), ornithine (Orn),Nα-Methylornithine (Nα-MeOrn or NMeOrn), sarcosine (Sar), homolysine(hLys or hK), homoarginine (hArg or hR), homoglutamine (hQ),Nα-methylarginine (NMeR), Nα-methylleucine (Nα-MeL or NMeL),N-methylhomolysine (NMeHoK), Nα-methylglutamine (NMeQ), norleucine(Nle), norvaline (Nva), 1,2,3,4-tetrahydroisoquinoline (Tic),Octahydroindole-2-carboxylic acid (Oic), 3-(1-naphthyl)alanine (1-Nal),3-(2-naphthyl)alanine (2-Nal), 1,2,3,4-tetrahydroisoquinoline (Tic),2-indanylglycine (IgI), para-iodophenylalanine (pI-Phe),para-aminophenylalanine (4AmP or 4-Amino-Phe), 4-guanidino phenylalanine(Guf), glycyllysine (abbreviated “K(Nε-glycyl)” or “K(glycyl)” or“K(gly)”), nitrophenylalanine (nitrophe), aminophenylalanine (aminopheor Amino-Phe), benzylphenylalanine (benzylphe), γ-carboxyglutamic acid(γ-carboxyglu), hydroxyproline (hydroxypro), p-carboxyl-phenylalanine(Cpa), α-aminoadipic acid (Aad), Nα-methyl valine (NMeVal), N-α-methylleucine (NMeLeu), Nα-methylnorleucine (NMeNle), cyclopentylglycine(Cpg), cyclohexylglycine (Chg), acetylarginine (acetylarg), α,β-diaminopropionoic acid (Dpr), α, γ-diaminobutyric acid (Dab),diaminopropionic acid (Dap), cyclohexylalanine (Cha),4-methyl-phenylalanine (MePhe), β, β-diphenyl-alanine (BiPhA),aminobutyric acid (Abu), 4-phenyl-phenylalanine (or biphenylalanine;4Bip), α-amino-isobutyric acid (Aib), beta-alanine, beta-aminopropionicacid, piperidinic acid, aminocaprioic acid, aminoheptanoic acid,aminopimelic acid, desmosine, diaminopimelic acid, N-ethylglycine,N-ethylaspargine, hydroxylysine, allo-hydroxylysine, isodesmosine,allo-isoleucine, N-methylglycine, N-methylisoleucine, N-methylvaline,4-hydroxyproline (Hyp), γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, ω-methylarginine, 4-Amino-O-PhthalicAcid (4APA), and other similar amino acids, and derivatized forms of anyof those specifically listed.

II. General Overview

Antigen-binding proteins that bind (i) β-Klotho; (ii) FGFR1c, FGFR2c,FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one ofFGFR1c, FGFR2c, FGFR3c, and FGFR4, are provided herein. A uniqueproperty of the antigen binding proteins disclosed herein is theagonistic nature of these proteins, specifically the ability to mimicthe in vivo effect of FGF21 and to induce FGF21-like signaling. Moreremarkably and specifically, some of the antigen binding proteinsdisclosed herein induce FGF21-like signaling in several in vitrocell-based assay, including the ELK-luciferase reporter assay of Example5 under the following conditions: (1) the binding to and activity of theFGF21 receptor is β-Klotho dependent; (2) the activity is selective toFGFR1c/βKlotho complex; (3) the binding to the FGFR1c/βKlotho triggersFGF21-like signaling pathways; and (4) the potency (EC50) is comparableto a wild-type FGF21 standard comprising the mature form of SEQ ID NO:2,as measured in the following cell-based assays: (1) the recombinantFGF21 receptor mediated luciferase-reporter cell assay of Example 5; (2)the ERK-phosphorylation in the recombinant FGF21 receptor mediated cellassay of Example 5; and (3) ERK-phosphorylation in human adipocytes asdescribed in more details in Example 7. The disclosed antigen bindingproteins, therefore, are expected to exhibit activities in vivo that areconsistent with the natural biological function of FGF21. This propertymakes the disclosed antigen binding proteins viable therapeutics for thetreatment of metabolic diseases such as type 2 diabetes, obesity,dyslipidemia, NASH, cardiovascular disease, metabolic syndrome andbroadly any disease or condition in which it is desirable to mimic oraugment the in vivo effects of FGF21.

In some embodiments of the present disclosure the antigen bindingproteins provided can comprise polypeptides into which one or morecomplementary determining regions (CDRs) can be embedded and/or joined.In such antigen binding proteins, the CDRs can be embedded into a“framework” region, which orients the CDR(s) such that the properantigen binding properties of the CDR(s) is achieved. In general, suchantigen binding proteins that are provided can facilitate or enhance theinteraction between FGFR1c and β-Klotho, and can substantially induceFGF21-like signaling.

Certain antigen binding proteins described herein are antibodies or arederived from antibodies. In certain embodiments, the polypeptidestructure of the antigen binding proteins is based on antibodies,including, but not limited to, monoclonal antibodies, bispecificantibodies, minibodies, domain antibodies, synthetic antibodies(sometimes referred to herein as “antibody mimetics”), chimericantibodies, humanized antibodies, human antibodies, antibody fusions(sometimes referred to herein as “antibody conjugates”), hemibodies andfragments thereof. The various structures are further described hereinbelow.

The antigen binding proteins provided herein have been demonstrated tobind to (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) acomplex comprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, andFGFR4, and particularly to (i) human β-Klotho; (ii) human FGFR1c, humanFGFR2c, human FGFR3c or human FGFR4; or (iii) a complex comprising humanβ-Klotho and one of human FGFR1c, human FGFR2c, human FGFR3c, and humanFGFR4. As described and shown in the Examples presented herein, basedthe Western blot results, commercially-available anti-β-Klotho oranti-FGFR1c antibodies bind to denatured β-Klotho or FGFR1c whereas theantigen binding protein (agonistic antibodies) do not. Conversely, theprovided antigen binding proteins recognize the native structure of theFGFR1c and β-Klotho on the cell surface whereas the commercialantibodies do not, based on the FACS results provided. See Example 9.The antigen binding proteins that are provided therefore mimic thenatural in vivo biological activity of FGF21. As a consequence, theantigen binding proteins provided herein are capable of activatingFGF21-like signaling activity. In particular, the disclosed antigenbinding proteins can have one or more of the following activities invivo: induction of FGF21-like signal transduction pathways, loweringblood glucose levels, lowering circulating lipid levels, improvingmetabolic parameters and other physiological effects induced in vivo bythe formation of the ternary complex of FGFR1c, β-Klotho and FGF21, forexample in conditions such as type 2 diabetes, obesity, dyslipidemia,NASH, cardiovascular disease, and metabolic syndrome.

The antigen binding proteins that specifically bind to (i) β-Klotho;(ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprisingβ-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 that are disclosedherein have a variety of utilities. Some of the antigen bindingproteins, for instance, are useful in specific binding assays, in theaffinity purification of (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c orFGFR4; or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c,FGFR3c, and FGFR4, including the human forms of these disclosedproteins, and in screening assays to identify other agonists ofFGF21-like signaling activity.

The antigen binding proteins that specifically bind (i) β-Klotho; (ii)FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klothoand one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 that are disclosed hereincan be used in a variety of treatment applications, as explained herein.For example, certain antigen binding proteins are useful for treatingconditions associated with FGF21-like signaling processes in a patient,such as reducing, alleviating, or treating type 2 diabetes, obesity,dyslipidemia, NASH, cardiovascular disease, and metabolic syndrome.Other uses for the antigen binding proteins include, for example,diagnosis of diseases or conditions associated with β-Klotho, FGFR1c,FGFR2c, FGFR3c, FGFR4 or FGF21, and screening assays to determine thepresence or absence of these molecules. Some of the antigen bindingproteins described herein can be useful in treating conditions, symptomsand/or the pathology associated with decreased FGF21-like signalingactivity. Exemplary conditions include, but are not limited to,diabetes, obesity, NASH and dyslipidemia.

FGF21

The antigen binding proteins disclosed herein induce FGF21-mediatedsignaling, as defined herein. In vivo, the mature form of FGF21 is theactive form of the molecule. The nucleotide sequence encoding fulllength FGF21 is provided; the nucleotides encoding the signal sequenceare underlined.

(SEQ ID NO: 1)ATG GAC TCG GAC GAG ACC GGG TTC GAG CAC TCA GGA CTG TGG GTT TCT GTGCTG GCT GGT CTT CTG CTG GGA GCC TGC CAG GCA CAC CCC ATC CCT GAC TCC AGTCCT CTC CTG CAA TTC GGG GGC CAA GTC CGG CAG CGG TAC CTC TAC ACA GAT GATGCC CAG CAG ACA GAA GCC CAC CTG GAG ATC AGG GAG GAT GGG ACG GTG GGG GGCGCT GCT GAC CAG AGC CCC GAA AGT CTC CTG CAG CTG AAA GCC TTG AAG CCG GGAGTT ATT CAA ATC TTG GGA GTC AAG ACA TCC AGG TTC CTG TGC CAG CGG CCA GATGGG GCC CTG TAT GGA TCG CTC CAC TTT GAC CCT GAG GCC TGC AGC TTC CGG GAGCTG CTT CTT GAG GAC GGA TAC AAT GTT TAC CAG TCC GAA GCC CAC GGC CTC CCGCTG CAC CTG CCA GGG AAC AAG TCC CCA CAC CGG GAC CCT GCA CCC CGA GGA CCAGCT CGC TTC CTG CCA CTA CCA GGC CTG CCC CCC GCA CCC CCG GAG CCA CCC GGAATC CTG GCC CCC CAG CCC CCC GAT GTG GGC TCC TCG GAC CCT CTG AGC ATG GTGGGA CCT TCC CAG GGC CGA AGC CCC AGC TAC GCT TCC TGA

The amino acid sequence of full length FGF21 is provided; the aminoacids that make up the signal sequence are underlined:

(SEQ ID NO: 2) M D S D E T G F E H S G L W V S V L A G L L L G A C Q A HP I P D S S P L L Q F G G Q V R Q R Y L Y T D D A Q Q T E A H L E I R ED G T V G G A A D Q S P E S L L Q L K A L K P G V I Q I L G V K T S R FL C Q R P D G A L Y G S L H F D P E A C S F R E L L L E D G Y N V Y Q SE A H G L P L H L P G N K S P H R D P A P R G P A R F L P L P G L P P AP P E P P G I L A P Q P P D V G S S D P L S M V G P S Q G R S P S Y A SFGFR1c

The antigen binding proteins disclosed herein bind to FGFR1c, inparticular human FGFR1c, when associated with β-Klotho. The nucleotidesequence encoding human FGFR1c (GenBank™ Accession Number NM_(—)023110)is provided:

(SEQ ID NO: 3) ATGTGGAGCTGGAAGTGCCTCCTCTTCTGGGCTGTGCTGGTCACAGCCACACTCTGCACCGCTAGGCCGTCCCCGACCTTGCCTGAACAAGCCCAGCCCTGGGGAGCCCCTGTGGAAGTGGAGTCCTTCCTGGTCCACCCCGGTGACCTGCTGCAGCTTCGCTGTCGGCTGCGGGACGATGTGCAGAGCATCAACTGGCTGCGGGACGGGGTGCAGCTGGCGGAAAGCAACCGCACCCGCATCACAGGGGAGGAGGTGGAGGTGCAGGACTCCGTGCCCGCAGACTCCGGCCTCTATGCTTGCGTAACCAGCAGCCCCTCGGGCAGTGACACCACCTACTTCTCCGTCAATGTTTCAGATGCTCTCCCCTCCTCGGAGGATGATGATGATGATGATGACTCCTCTTCAGAGGAGAAAGAAACAGATAACACCAAACCAAACCGTATGCCCGTAGCTCCATATTGGACATCACCAGAAAAGATGGAAAAGAAATTGCATGCAGTGCCGGCTGCCAAGACAGTGAAGTTCAAATGCCCTTCCAGTGGGACACCAAACCCAACACTGCGCTGGTTGAAAAATGGCAAAGAATTCAAACCTGACCACAGAATTGGAGGCTACAAGGTCCGTTATGCCACCTGGAGCATCATAATGGACTCTGTGGTGCCCTCTGACAAGGGCAACTACACCTGCATTGTGGAGAATGAGTACGGCAGCATCAACCACACATACCAGCTGGATGTCGTGGAGCGGTCCCCTCACCGGCCCATCCTGCAAGCAGGGTTGCCCGCCAACAAAACAGTGGCCCTGGGTAGCAACGTGGAGTTCATGTGTAAGGTGTACAGTGACCCGCAGCCGCACATCCAGTGGCTAAAGCACATCGAGGTGAATGGGAGCAAGATTGGCCCAGACAACCTGCCTTATGTCCAGATCTTGAAGACTGCTGGAGTTAATACCACCGACAAAGAGATGGAGGTGCTTCACTTAAGAAATGTCTCCTTTGAGGACGCAGGGGAGTATACGTGCTTGGCGGGTAACTCTATCGGACTCTCCCATCACTCTGCATGGTTGACCGTTCTGGAAGCCCTGGAAGAGAGGCCGGCAGTGATGACCTCGCCCCTGTACCTGGAGATCATCATCTATTGCACAGGGGCCTTCCTCATCTCCTGCATGGTGGGGTCGGTCATCGTCTACAAGATGAAGAGTGGTACCAAGAAGAGTGACTTCCACAGCCAGATGGCTGTGCACAAGCTGGCCAAGAGCATCCCTCTGCGCAGACAGGTAACAGTGTCTGCTGACTCCAGTGCATCCATGAACTCTGGGGTTCTTCTGGTTCGGCCATCACGGCTCTCCTCCAGTGGGACTCCCATGCTAGCAGGGGTCTCTGAGTATGAGCTTCCCGAAGACCCTCGCTGGGAGCTGCCTCGGGACAGACTGGTCTTAGGCAAACCCCTGGGAGAGGGCTGCTTTGGGCAGGTGGTGTTGGCAGAGGCTATCGGGCTGGACAAGGACAAACCCAACCGTGTGACCAAAGTGGCTGTGAAGATGTTGAAGTCGGACGCAACAGAGAAAGACTTGTCAGACCTGATCTCAGAAATGGAGATGATGAAGATGATCGGGAAGCATAAGAATATCATCAACCTGCTGGGGGCCTGCACGCAGGATGGTCCCTTGTATGTCATCGTGGAGTATGCCTCCAAGGGCAACCTGCGGGAGTACCTGCAGGCCCGGAGGCCCCCAGGGCTGGAATACTGCTACAACCCCAGCCACAACCCAGAGGAGCAGCTCTCCTCCAAGGACCTGGTGTCCTGCGCCTACCAGGTGGCCCGAGGCATGGAGTATCTGGCCTCCAAGAAGTGCATACACCGAGACCTGGCAGCCAGGAATGTCCTGGTGACAGAGGACAATGTGATGAAGATAGCAGACTTTGGCCTCGCACGGGACATTCACCACATCGACTACTATAAAAAGACAACCAACGGCCGACTGCCTGTGAAGTGGATGGCACCCGAGGCATTATTTGACCGGATCTACACCCACCAGAGTGATGTGTGGTCTTTCGGGGTGCTCCTGTGGGAGATCTTCACTCTGGGCGGCTCCCCATACCCCGGTGTGCCTGTGGAGGAACTTTTCAAGCTGCTGAAGGAGGGTCACCGCATGGACAAGCCCAGTAACTGCACCAACGAGCTGTACATGATGATGCGGGACTGCTGGCATGCAGTGCCCTCACAGAGACCCACCTTCAAGCAGCTGGTGGAAGACCTGGACCGCATCGTGGCCTTGACCTCCAACCAGGAGTACCTGGACCTGTCCATGCCCCTGGACCAGTACTCCCCCAGCTTTCCCGACACCCGGAGCTCTACGTGCTCCTCAGGGGAGGATTCCGTCTTCTCTCATGAGCCGCTGCCCGAGGAGCCCTGCCTGCCCCGACACCCAGCCCAGCTTGCCAATGGCGGACTCAAACGCCGCTGA.

The amino acid sequence of human FGFR1c (GenBank™ Accession NumberNP_(—)075598)is provided:

(SEQ ID NO: 4) MWSWKCLLFWAVLVTATLCTARPSPTLPEQAQPWGAPVEVESFLVHPGDLLQLRCRLRDDVQSINWLRDGVQLAESNRTRITGEEVEVQDSVPADSGLYACVTSSPSGSDTTYFSVNVSDALPSSEDDDDDDDSSSEEKETDNTKPNRMPVAPYWTSPEKMEKKLHAVPAAKTVKFKCPSSGTPNPTLRWLKNGKEFKPDHRIGGYKVRYATWSIIMDSVVPSDKGNYTCIVENEYGSINHTYQLDVVERSPHRPILQAGLPANKTVALGSNVEFMCKVYSDPQPHIQWLKHIEVNGSKIGPDNLPYVQILKTAGVNTTDKEMEVLHLRNVSFEDAGEYTCLAGNSIGLSHHSAWLTVLEALEERPAVMTSPLYLEIIIYCTGAFLISCMVGSVIVYKMKSGTKKSDFHSQMAVHKLAKSIPLRRQVTVSADSSASMNSGVLLVRPSRLSSSGTPMLAGVSEYELPEDPRWELPRDRLVLGKPLGEGCFGQVVLAEAIGLDKDKPNRVTKVAVKMLKSDATEKDLSDLISEMEMMKMIGKHKNIINLLGACTQDGPLYVIVEYASKGNLREYLQARRPPGLEYCYNPSHNPEEQLSSKDLVSCAYQVARGMEYLASKKCIHRDLAARNVLVTEDNVMKIADFGLARDIHHIDYYKKTTNGRLPVKWMAPEALFDRIYTHQSDVWSFGVLLWEIFTLGGSPYPGVPVEELFKLLKEGHRMDKPSNCTNELYMMMRDCWHAVPSQRPTFKQLVEDLDRIVALTSNQEYLDLSMPLDQYSPSFPDTRSSTCSSGEDSVFSHEPLPEEPCLPRHPAQLANGGLKRR.

The antigen binding proteins described herein bind the extracellularportion of FGFR1c. An example of an extracellular region of FGFR1c is:

(SEQ ID NO: 5) MWSWKCLLFWAVLVTATLCTARPSPTLPEQAQPWGAPVEVESFLVHPGDLLQLRCRLRDDVQSINWLRDGVQLAESNRTRITGEEVEVQDSVPADSGLYACVTSSPSGSDTTYFSVNVSDALPSSEDDDDDDDSSSEEKETDNTKPNRMPVAPYWTSPEKMEKKLHAVPAAKTVKFKCPSSGTPNPTLRWLKNGKEFKPDHRIGGYKVRYATWSIIMDSVVPSDKGNYTCIVENEYGSINHTYQLDVVERSPHRPILQAGLPANKTVALGSNVEFMCKVYSDPQPHIQWLKHIEVNGSKIGPDNLPYVQILKTAGVNTTDKEMEVLHLRNVSFEDAGEYTCLAGNSIGLSHHSAWLTVLEALEERPAVMTSPLY.

As described herein, FGFR1c proteins can also include fragments. As usedherein, the terms are used interchangeably to mean a receptor, inparticular and unless otherwise specified, a human receptor, that uponassociation with β-Klotho and FGF21 induces FGF21-like signalingactivity.

The term FGFR1c also includes post-translational modifications of theFGFR1c amino acid sequence, for example, possible N-linked glycosylationsites. Thus, the antigen binding proteins can bind to or be generatedfrom proteins glycosylated at one or more of the positions.

β-Klotho

The antigen binding proteins disclosed herein bind to β-Klotho, inparticular human β-Klotho. The nucleotide sequence encoding humanβ-Klotho (GenBank™ Accession Number NM_(—)175737) is provided:

(SEQ ID NO: 6) ATGAAGCCAGGCTGTGCGGCAGGATCTCCAGGGAATGAATGGATTTTCTTCAGCACTGATGAAATAACCACACGCTATAGGAATACAATGTCCAACGGGGGATTGCAAAGATCTGTCATCCTGTCAGCACTTATTCTGCTACGAGCTGTTACTGGATTCTCTGGAGATGGAAGAGCTATATGGTCTAAAAATCCTAATTTTACTCCGGTAAATGAAAGTCAGCTGTTTCTCTATGACACTTTCCCTAAAAACTTTTTCTGGGGTATTGGGACTGGAGCATTGCAAGTGGAAGGGAGTTGGAAGAAGGATGGAAAAGGACCTTCTATATGGGATCATTTCATCCACACACACCTTAAAAATGTCAGCAGCACGAATGGTTCCAGTGACAGTTATATTTTTCTGGAAAAAGACTTATCAGCCCTGGATTTTATAGGAGTTTCTTTTTATCAATTTTCAATTTCCTGGCCAAGGCTTTTCCCCGATGGAATAGTAACAGTTGCCAACGCAAAAGGTCTGCAGTACTACAGTACTCTTCTGGACGCTCTAGTGCTTAGAAACATTGAACCTATAGTTACTTTATACCACTGGGATTTGCCTTTGGCACTACAAGAAAAATATGGGGGGTGGAAAAATGATACCATAATAGATATCTTCAATGACTATGCCACATACTGTTTCCAGATGTTTGGGGACCGTGTCAAATATTGGATTACAATTCACAACCCATATCTAGTGGCTTGGCATGGGTATGGGACAGGTATGCATGCCCCTGGAGAGAAGGGAAATTTAGCAGCTGTCTACACTGTGGGACACAACTTGATCAAGGCTCACTCGAAAGTTTGGCATAACTACAACACACATTTCCGCCCACATCAGAAGGGTTGGTTATCGATCACGTTGGGATCTCATTGGATCGAGCCAAACCGGTCGGAAAACACGATGGATATATTCAAATGTCAACAATCCATGGTTTCTGTGCTTGGATGGTTTGCCAACCCTATCCATGGGGATGGCGACTATCCAGAGGGGATGAGAAAGAAGTTGTTCTCCGTTCTACCCATTTTCTCTGAAGCAGAGAAGCATGAGATGAGAGGCACAGCTGATTTCTTTGCCTTTTCTTTTGGACCCAACAACTTCAAGCCCCTAAACACCATGGCTAAAATGGGACAAAATGTTTCACTTAATTTAAGAGAAGCGCTGAACTGGATTAAACTGGAATACAACAACCCTCGAATCTTGATTGCTGAGAATGGCTGGTTCACAGACAGTCGTGTGAAAACAGAAGACACCACGGCCATCTACATGATGAAGAATTTCCTCAGCCAGGTGCTTCAAGCAATAAGGTTAGATGAAATACGAGTGTTTGGTTATACTGCCTGGTCTCTCCTGGATGGCTTTGAATGGCAGGATGCTTACACCATCCGCCGAGGATTATTTTATGTGGATTTTAACAGTAAACAGAAAGAGCGGAAACCTAAGTCTTCAGCACACTACTACAAACAGATCATACGAGAAAATGGTTTTTCTTTAAAAGAGTCCACGCCAGATGTGCAGGGCCAGTTTCCCTGTGACTTCTCCTGGGGTGTCACTGAATCTGTTCTTAAGCCCGAGTCTGTGGCTTCGTCCCCACAGTTCAGCGATCCTCATCTGTACGTGTGGAACGCCACTGGCAACAGACTGTTGCACCGAGTGGAAGGGGTGAGGCTGAAAACACGACCCGCTCAATGCACAGATTTTGTAAACATCAAAAAACAACTTGAGATGTTGGCAAGAATGAAAGTCACCCACTACCGGTTTGCTCTGGATTGGGCCTCGGTCCTTCCCACTGGCAACCTGTCCGCGGTGAACCGACAGGCCCTGAGGTACTACAGGTGCGTGGTCAGTGAGGGGCTGAAGCTTGGCATCTCCGCGATGGTCACCCTGTATTATCCGACCCACGCCCACCTAGGCCTCCCCGAGCCTCTGTTGCATGCCGACGGGTGGCTGAACCCATCGACGGCCGAGGCCTTCCAGGCCTACGCTGGGCTGTGCTTCCAGGAGCTGGGGGACCTGGTGAAGCTCTGGATCACCATCAACGAGCCTAACCGGCTAAGTGACATCTACAACCGCTCTGGCAACGACACCTACGGGGCGGCGCACAACCTGCTGGTGGCCCACGCCCTGGCCTGGCGCCTCTACGACCGGCAGTTCAGGCCCTCACAGCGCGGGGCCGTGTCGCTGTCGCTGCACGCGGACTGGGCGGAACCCGCCAACCCCTATGCTGACTCGCACTGGAGGGCGGCCGAGCGCTTCCTGCAGTTCGAGATCGCCTGGTTCGCCGAGCCGCTCTTCAAGACCGGGGACTACCCCGCGGCCATGAGGGAATACATTGCCTCCAAGCACCGACGGGGGCTTTCCAGCTCGGCCCTGCCGCGCCTCACCGAGGCCGAAAGGAGGCTGCTCAAGGGCACGGTCGACTTCTGCGCGCTCAACCACTTCACCACTAGGTTCGTGATGCACGAGCAGCTGGCCGGCAGCCGCTACGACTCGGACAGGGACATCCAGTTTCTGCAGGACATCACCCGCCTGAGCTCCCCCACGCGCCTGGCTGTGATTCCCTGGGGGGTGCGCAAGCTGCTGCGGTGGGTCCGGAGGAACTACGGCGACATGGACATTTACATCACCGCCAGTGGCATCGACGACCAGGCTCTGGAGGATGACCGGCTCCGGAAGTACTACCTAGGGAAGTACCTTCAGGAGGTGCTGAAAGCATACCTGATTGATAAAGTCAGAATCAAAGGCTATTATGCATTCAAACTGGCTGAAGAGAAATCTAAACCCAGATTTGGATTCTTCACATCTGATTTTAAAGCTAAATCCTCAATACAATTTTACAACAAAGTGATCAGCAGCAGGGGCTTCCCTTTTGAGAACAGTAGTTCTAGATGCAGTCAGACCCAAGAAAATACAGAGTGCACTGTCTGCTTATTCCTTGTGCAGAAGAAACCACTGATATTCCTGGGTTGTTGCTTCTTCTCCACCCTGGTTCTACTCTTATCAATTGCCATTTTTCAAAGGCAGAAGAGAAGAAAGTTTTGGAAAGCAAAAAACTTACAACACATACCATTAAAGAAAGGC AAGAGAGTTGTTAGCTAA.

The amino acid sequence of full length human β-Klotho (GenBank™Accession Number NP_(—)783864) is provided:

(SEQ ID NO: 7) MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAVTGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSWKKDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQFSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPLALQEKYGGWKNDTIIDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGYGTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITLGSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFSVLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREALNWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLDEIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYKQIIRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLGLPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYNRSGNDTYGAAHNLLVAHALAWRLYDRQFRPSQRGAVSLSLHADWAEPANPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFLGCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRVVS.

The antigen binding proteins described herein bind the extracellularportion of β-Klotho. An example of an extracellular region of β-Klothois:

(SEQ ID NO: 8.) MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAVTGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSWKKDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQFSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPLALQEKYGGWKNDTIIDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGYGTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITLGSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFSVLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREALNWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLDEIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYKQIIRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLGLPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYNRSGNDTYGAAHNLLVAHALAWRLYDRQFRPSQRGAVSLSLHADWAEPANPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKP

The murine form of β-Klotho, and fragments and subsequences thereof, canbe of use in studying and/or constructing the molecules provided herein.The nucleotide sequence encoding murine β-Klotho (GenBank™ AccessionNumber NM_(—)031180) is provided:

(SEQ ID NO: 469) ATGAAGACAGGCTGTGCAGCAGGGTCTCCGGGGAATGAATGGATTTTCTTCAGCTCTGATGAAAGAAACACACGCTCTAGGAAAACAATGTCCAACAGGGCACTGCAAAGATCTGCCGTGCTGTCTGCGTTTGTTCTGCTGCGAGCTGTTACCGGCTTCTCCGGAGACGGGAAAGCAATATGGGATAAAAAACAGTACGTGAGTCCGGTAAACCCAAGTCAGCTGTTCCTCTATGACACTTTCCCTAAAAACTTTTCCTGGGGCGTTGGGACCGGAGCATTTCAAGTGGAAGGGAGTTGGAAGACAGATGGAAGAGGACCCTCGATCTGGGATCGGTACGTCTACTCACACCTGAGAGGTGTCAACGGCACAGACAGATCCACTGACAGTTACATCTTTCTGGAAAAAGACTTGTTGGCTCTGGATTTTTTAGGAGTTTCTTTTTATCAGTTCTCAATCTCCTGGCCACGGTTGTTTCCCAATGGAACAGTAGCAGCAGTGAATGCGCAAGGTCTCCGGTACTACCGTGCACTTCTGGACTCGCTGGTACTTAGGAATATCGAGCCCATTGTTACCTTGTACCATTGGGATTTGCCTCTGACGCTCCAGGAAGAATATGGGGGCTGGAAAAATGCAACTATGATAGATCTCTTCAACGACTATGCCACATACTGCTTCCAGACCTTTGGAGACCGTGTCAAATATTGGATTACAATTCACAACCCTTACCTTGTTGCTTGGCATGGGTTTGGCACAGGTATGCATGCACCAGGAGAGAAGGGAAATTTAACAGCTGTCTACACTGTGGGACACAACCTGATCAAGGCACATTCGAAAGTGTGGCATAACTACGACAAAAACTTCCGCCCTCATCAGAAGGGTTGGCTCTCCATCACCTTGGGGTCCCATTGGATAGAGCCAAACAGAACAGACAACATGGAGGACGTGATCAACTGCCAGCACTCCATGTCCTCTGTGCTTGGATGGTTCGCCAACCCCATCCACGGGGACGGCGACTACCCTGAGTTCATGAAGACGGGCGCCATGATCCCCGAGTTCTCTGAGGCAGAGAAGGAGGAGGTGAGGGGCACGGCTGATTTCTTTGCCTTTTCCTTCGGGCCCAACAACTTCAGGCCCTCAAACACCGTGGTGAAAATGGGACAAAATGTATCACTCAACTTAAGGCAGGTGCTGAACTGGATTAAACTGGAATACGATGACCCTCAAATCTTGATTTCGGAGAACGGCTGGTTCACAGATAGCTATATAAAGACAGAGGACACCACGGCCATCTACATGATGAAGAATTTCCTAAACCAGGTTCTTCAAGCAATAAAATTTGATGAAATCCGCGTGTTTGGTTATACGGCCTGGACTCTCCTGGATGGCTTTGAGTGGCAGGATGCCTATACGACCCGACGAGGGCTGTTTTATGTGGACTTTAACAGTGAGCAGAAAGAGAGGAAACCCAAGTCCTCGGCTCATTACTACAAGCAGATCATACAAGACAACGGCTTCCCTTTGAAAGAGTCCACGCCAGACATGAAGGGTCGGTTCCCCTGTGATTTCTCTTGGGGAGTCACTGAGTCTGTTCTTAAGCCCGAGTTTACGGTCTCCTCCCCGCAGTTTACCGATCCTCACCTGTATGTGTGGAATGTCACTGGCAACAGATTGCTCTACCGAGTGGAAGGGGTAAGGCTGAAAACAAGACCATCCCAGTGCACAGATTATGTGAGCATCAAAAAACGAGTTGAAATGTTGGCAAAAATGAAAGTCACCCACTACCAGTTTGCTCTGGACTGGACCTCTATCCTTCCCACTGGCAATCTGTCCAAAGTTAACAGACAAGTGTTAAGGTACTATAGGTGTGTGGTGAGCGAAGGACTGAAGCTGGGCGTCTTCCCCATGGTGACGTTGTACCACCCAACCCACTCCCATCTCGGCCTCCCCCTGCCACTTCTGAGCAGTGGGGGGTGGCTAAACATGAACACAGCCAAGGCCTTCCAGGACTACGCTGAGCTGTGCTTCCGGGAGTTGGGGGACTTGGTGAAGCTCTGGATCACCATCAATGAGCCTAACAGGCTGAGTGACATGTACAACCGCACGAGTAATGACACCTACCGTGCAGCCCACAACCTGATGATCGCCCATGCCCAGGTCTGGCACCTCTATGATAGGCAGTATAGGCCGGTCCAGCATGGGGCTGTGTCGCTGTCCTTACATTGCGACTGGGCAGAACCTGCCAACCCCTTTGTGGATTCACACTGGAAGGCAGCCGAGCGCTTCCTCCAGTTTGAGATCGCCTGGTTTGCAGATCCGCTCTTCAAGACTGGCGACTATCCATCGGTTATGAAGGAATACATCGCCTCCAAGAACCAGCGAGGGCTGTCTAGCTCAGTCCTGCCGCGCTTCACCGCGAAGGAGAGCAGGCTGGTGAAGGGTACCGTCGACTTCTACGCACTGAACCACTTCACTACGAGGTTCGTGATACACAAGCAGCTGAACACCAACCGCTCAGTTGCAGACAGGGACGTCCAGTTCCTGCAGGACATCACCCGCCTAAGCTCGCCCAGCCGCCTGGCTGTAACACCCTGGGGAGTGCGCAAGCTCCTTGCGTGGATCCGGAGGAACTACAGAGACAGGGATATCTACATCACAGCCAATGGCATCGATGACCTGGCTCTAGAGGATGATCAGATCCGAAAGTACTACTTGGAGAAGTATGTCCAGGAGGCTCTGAAAGCATATCTCATTGACAAGGTCAAAATCAAAGGCTACTATGCATTCAAACTGACTGAAGAGAAATCTAAGCCTAGATTTGGATTTTTCACCTCTGACTTCAGAGCTAAGTCCTCTGTCCAGTTTTACAGCAAGCTGATCAGCAGCAGTGGCCTCCCCGCTGAGAACAGAAGTCCTGCGTGTGGTCAGCCTGCGGAAGACACAGACTGCACCATTTGCTCATTTCTCGTGGAGAAGAAACCACTCATCTTCTTCGGTTGCTGCTTCATCTCCACTCTGGCTGTACTGCTATCCATCACCGTTTTTCATCATCAAAAGAGAAGAAAATTCCAGAAAGCAAGGAACTTACAAAATATACCATTGAAGAAAGGCCACAGCAGAGTTTTCAGCTAA

The amino acid sequence of full length murine β-Klotho (GenBank™Accession Number NP_(—)112457) is provided:

(SEQ ID NO: 468) MKTGCAAGSPGNEWIFFSSDERNTRSRKTMSNRALQRSAVLSAFVLLRAVTGFSGDGKAIWDKKQYVSPVNPSQLFLYDTFPKNFSWGVGTGAFQVEGSWKTDGRGPSIWDRYVYSHLRGVNGTDRSTDSYIFLEKDLLALDFLGVSFYQFSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEPIVTLYHWDLPLTLQEEYGGWKNATMIDLFNDYATYCFQTFGDRVKYWITIHNPYLVAWHGFGTGMHAPGEKGNLTAVYTVGHNLIKAHSKVWHNYDKNFRPHQKGWLSITLGSHWIEPNRTDNMEDVINCQHSMSSVLGWFANPIHGDGDYPEFMKTGAMIPEFSEAEKEEVRGTADFFAFSFGPNNFRPSNTVVKMGQNVSLNLRQVLNWIKLEYDDPQILISENGWFTDSYIKTEDTTAIYMMKNFLNQVLQAIKFDEIRVFGYTAWTLLDGFEWQDAYTTRRGLFYVDFNSEQKERKPKSSAHYYKQIIQDNGFPLKESTPDMKGRFPCDFSWGVTESVLKPEFTVSSPQFTDPHLYVWNVTGNRLLYRVEGVRLKTRPSQCTDYVSIKKRVEMLAKMKVTHYQFALDWTSILPTGNLSKVNRQVLRYYRCVVSEGLKLGVFPMVTLYHPTHSHLGLPLPLLSSGGWLNMNTAKAFQDYAELCFRELGDLVKLWITINEPNRLSDMYNRTSNDTYRAAHNLMIAHAQVWHLYDRQYRPVQHGAVSLSLHCDWAEPANPFVDSHWKAAERFLQFEIAWFADPLFKTGDYPSVMKEYIASKNQRGLSSSVLPRFTAKESRLVKGTVDFYALNHFTTRFVIHKQLNTNRSVADRDVQFLQDITRLSSPSRLAVTPWGVRKLLAWIRRNYRDRDIYITANGIDDLALEDDQIRKYYLEKYVQEALKAYLIDKVKIKGYYAFKLTEEKSKPRFGFFTSDFRAKSSVQFYSKLISSSGLPAENRSPACGQPAEDTDCTICSFLVEKKPLIFFGCCFISTLAVLLSITVFHHQKRRKFQKARNLQNIPLKKGH SRVFS

As described herein, β-Klotho proteins can also include fragments. Asused herein, the terms are used interchangeably to mean a co-receptor,in particular and unless otherwise specified, a human co-receptor, thatupon association with FGFR1c and FGF21 induces FGF21-like signalingactivity.

The term β-Klotho also includes post-translational modifications of theβ-Klotho amino acid sequence, for example, possible N-linkedglycosylation sites. Thus, the antigen binding proteins can bind to orbe generated from proteins glycosylated at one or more of the positions.

Antigen Binding Proteins that Specifically Bind One or More of β-Klotho,FGFR1c, FGFR2c, FGFR3c, FGFR4c

A variety of selective binding agents useful for modulating FGF21-likesignaling are provided. These agents include, for instance, antigenbinding proteins that contain an antigen binding domain (e.g., singlechain antibodies, domain antibodies, hemibodies, immunoadhesions, andpolypeptides with an antigen binding region) and specifically bind toFGFR1c, β-Klotho or both FGFR1c and β-Klotho, in particular human FGFR1cand human β-Klotho. Some of the agents, for example, are useful inmimicking the signaling effect generated in vivo by the association ofFGFR1c with β-Klotho and with FGF21, and can thus be used to enhance ormodulate one or more activities associated with FGF21-like signaling.

In general, the antigen binding proteins that are provided typicallycomprise one or more CDRs as described herein (e.g., 1, 2, 3, 4, 5 or 6CDRs). In some embodiments the antigen binding proteins are naturallyexpressed by clones, while in other embodiments, the antigen bindingprotein can comprise (a) a polypeptide framework structure and (b) oneor more CDRs that are inserted into and/or joined to the polypeptideframework structure. In some of these embodiments a CDR forms acomponent of a heavy or light chains expressed by the clones describedherein; in other embodiments a CDR can be inserted into a framework inwhich the CDR is not naturally expressed. A polypeptide frameworkstructure can take a variety of different forms. For example, apolypeptide framework structure can be, or comprise, the framework of anaturally occurring antibody, or fragment or variant thereof, or it canbe completely synthetic in nature. Examples of various antigen bindingprotein structures are further described below.

In some embodiments in which the antigen binding protein comprises (a) apolypeptide framework structure and (b) one or more CDRs that areinserted into and/or joined to the polypeptide framework structure, thepolypeptide framework structure of an antigen binding protein is anantibody or is derived from an antibody, including, but not limited to,monoclonal antibodies, bispecific antibodies, minibodies, domainantibodies, synthetic antibodies (sometimes referred to herein as“antibody mimetics”), chimeric antibodies, humanized antibodies,antibody fusions (sometimes referred to as “antibody conjugates”), andportions or fragments of each, respectively. In some instances, theantigen binding protein is an immunological fragment of an antibody(e.g., a Fab, a Fab′, a F(ab′)₂, or a scFv).

Certain of the antigen binding proteins as provided herein specificallybind to (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) acomplex comprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, andFGFR4, including the human forms of these proteins. In one embodiment,an antigen binding protein specifically binds to both human FGFR1ccomprising the amino acid sequence of SEQ ID NO:5, and human β-Klothocomprising the amino acid sequence of SEQ ID NO:8, and in anotherembodiment an antigen binding protein specifically binds to both humanFGFR1c comprising the amino acid sequence of SEQ ID NO:5 and humanβ-Klotho having the amino acid sequence of SEQ ID NO:8 and inducesFGF21-like signaling. Thus, an antigen binding protein can, but neednot, induce FGF21-like signaling.

Antigen Binding Protein Structure

Some of the antigen binding proteins that specifically bind (i)β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4,including the human forms of these proteins that are provided hereinhave a structure typically associated with naturally occurringantibodies. The structural units of these antibodies typically compriseone or more tetramers, each composed of two identical couplets ofpolypeptide chains, though some species of mammals also produceantibodies having only a single heavy chain. In a typical antibody, eachpair or couplet includes one full-length “light” chain (in certainembodiments, about 25 kDa) and one full-length “heavy” chain (in certainembodiments, about 50-70 kDa). Each individual immunoglobulin chain iscomposed of several “immunoglobulin domains,” each consisting of roughly90 to 110 amino acids and expressing a characteristic folding pattern.These domains are the basic units of which antibody polypeptides arecomposed. The amino-terminal portion of each chain typically includes avariable domain that is responsible for antigen recognition. Thecarboxy-terminal portion is more conserved evolutionarily than the otherend of the chain and is referred to as the “constant region” or “Cregion”. Human light chains generally are classified as kappa (“κ”) andlambda (“λ”) light chains, and each of these contains one variabledomain and one constant domain. Heavy chains are typically classified asmu, delta, gamma, alpha, or epsilon chains, and these define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG hasseveral subtypes, including, but not limited to, IgG1, IgG2, IgG3, andIgG4. IgM subtypes include IgM, and IgM2. IgA subtypes include IgA1 andIgA2. In humans, the IgA and IgD isotypes contain four heavy chains andfour light chains; the IgG and IgE isotypes contain two heavy chains andtwo light chains; and the IgM isotype contains five heavy chains andfive light chains. The heavy chain C region typically comprises one ormore domains that can be responsible for effector function. The numberof heavy chain constant region domains will depend on the isotype. IgGheavy chains, for example, each contain three C region domains known asC_(H)1, C_(H)2 and C_(H)3. The antibodies that are provided can have anyof these isotypes and subtypes. In certain embodiments, an antigenbinding protein that specifically binds one or more of (i) β-Klotho;(ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprisingβ-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 is an antibody ofthe IgG1, IgG2, or IgG4 subtype.

In full-length light and heavy chains, the variable and constant regionsare joined by a “J” region of about twelve or more amino acids, with theheavy chain also including a “D” region of about ten more amino acids.See, e.g., Fundamental Immunology, 2nd ed., Ch. 7 (Paul, W., ed.) 1989,New York: Raven Press (hereby incorporated by reference in its entiretyfor all purposes). The variable regions of each light/heavy chain pairtypically form the antigen binding site.

One example of an IgG2 heavy constant domain of an exemplary monoclonalantibody that specifically binds (i) β-Klotho; (ii) FGFR1c, FGFR2c,FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one ofFGFR1c, FGFR2c, FGFR3c, and FGFR4 has the amino acid sequence:

(SEQ ID NO: 9) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

One example of a kappa light constant domain of an exemplary monoclonalantibody that binds (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4;or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c,FGFR3c, and FGFR4 has the amino acid sequence:

(SEQ ID NO: 10) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC.

One example of a lambda light constant domain of an exemplary monoclonalantibody that binds (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4;or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c,FGFR3c, and FGFR4 has the amino acid sequence:

(SEQ ID NO: 11) GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKT VAPTECS

Variable regions of immunoglobulin chains generally exhibit the sameoverall structure, comprising relatively conserved framework regions(FR) joined by three hypervariable regions, more often called“complementarity determining regions” or CDRs. The CDRs from the twochains of each heavy chain/light chain pair mentioned above typicallyare aligned by the framework regions to form a structure that bindsspecifically with a specific epitope on the target protein (e.g., (i)β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4). FromN-terminal to C-terminal, naturally-occurring light and heavy chainvariable regions both typically conform with the following order ofthese elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. A numberingsystem has been devised for assigning numbers to amino acids that occupypositions in each of these domains. This numbering system is defined inKabat Sequences of Proteins of Immunological Interest (1987 and 1991,NIH, Bethesda, Md.). As desired, the CDRs can also be redefinedaccording an alternative nomenclature scheme, such as that of Chothia(see Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al.,1989, Nature 342:878-883 or Honegger & Pluckthun, 2001, J. Mol. Biol.309:657-670.

The various heavy chain and light chain variable regions of antigenbinding proteins provided herein are depicted in Table 2. Each of thesevariable regions can be attached to the above heavy and light chainconstant regions to form a complete antibody heavy and light chain,respectively. Further, each of the so-generated heavy and light chainsequences can be combined to form a complete antibody structure. Itshould be understood that the heavy chain and light chain variableregions provided herein can also be attached to other constant domainshaving different sequences than the exemplary sequences listed above.

Specific examples of some of the full length light and heavy chains ofthe antibodies that are provided and their corresponding amino acidsequences are summarized in Tables 1A and 1B. Table 1A shows exemplarylight chain sequences, and Table 1B shows exemplary heavy chainsequences.

TABLE 1A Exemplary Antibody Light Chain Sequences SEQ ID Contained NO:Designation in Clone Amino Acid Sequence 12 L1  17C3SYVLTQPPSVSVAPGQTARITCGGNNIGSQSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATL TISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPG AVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 13 L2  22H5SYVLTQPPSVSVAPGQTARITCGGNNIGSQSVHWY QQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDNTSDHVVFGGGTKL TVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYA ASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 14 L3  16H7 SYVLTQPPSVSVAPGQTARITCGGNNIGSESVHWYQ 24H11QKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATL TISRVEAGDEADYYCQVWDGNSDHVVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYP GAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 15 L4  18G1EIVLTQSPGTLSLSPGERATLSCRASQNFDSSYLAWYQQKPGQAPRLLIYGTSSRATGIPDRFSGIGSGTDFTLTINRLEPEDFAMYYCQQYGGSPLTFGGGTEVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 16L5  17D8 EIVLTQSPGTLSLSPGERATLSCRASQSVSGNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSAPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 17L6  26H11 EIVLTQSPGTLSLSPGERATLSCRASQSVSGNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAMYYCQQYGSSPLTFGGGSKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 18 L7  12E4EIVLTQSPGTLSLSPGERATLSCRASQNFDSNYLAWYQQKPGQAPRLLIYGASSRATGIPDNFSGSGSGTDFTLTISRLEPEDFAMYYCQQYGSSPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 19L8  12C11 EIVLTQSPGTLSLSPGERATLSCRASQNFDSSSLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAMYYCQQCGSSPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 20L9  21H2 EIVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAWH 21B4QQKPGQGLRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSFTFGGGTRVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 21L10 18B11.1 DIVMTQSPLSLPVTPGEPASISCRSSQSLLYYNGFTYLDWFLQKPGQSPHLLIYLGSNRASGVPDRFSGSVSGTDFTLKISRVEAEDVGVYYCMQSLQTPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 22 L11 18B11.2EIVMTQSPATLSVSPGERATLSCRASQSVNSNLAWYQQKPGQAPRLLIYGVSTRATGIPARFSGSGSGTEFTLTIRSLQSEDFAVYYCQQYNNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 23L12 20D4 DIQLTQSPSSLSASIGDRVTITCRASQDIRYDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTVSSLQPEDFATYYCLQHNSYPLTFGGGTKVEIERTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 24L13 46D11 DIQMTQSPSSVSASVGDRVTITCRASQGISIWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANDFPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 25L14 40D2 DFVMTQTPLSLSVTPGQPASISCKSSQSLLQSDGKTYLYWYLQKPGQPPHLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSIQLPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 26 L15 37D3 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNFLDWYLQKPGQSPQLLIYLGSDRASGVPDRFSGSGSGTEFTLKISRVEAEDVGLYYCMQALQTPCSFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 27 L16 39F7EIVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSGSSPLTFGGGTEVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 28L17 39F11 EIVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQKPGQAPSLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSGSSPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 29L18 39G5 EIVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQKPGQAPRLLIYGASFRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSGSSPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

TABLE 1B Exemplary Antibody Heavy Chain Sequences SEQ Contained ID NO:Designation in Clone Sequence 30 H1  17C3QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMG VSWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARILLLGA YYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH QDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 31 H2  22H5QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMG VSWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARILLVGA YYYCGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH QDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 32 H3  16H7QVTLKESGPVLVKPTETLTLTCTVSGFSLNNARMG VSWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKSQVVLIMTNMDPVDTATYYCARSVVTGG YYYDGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH QDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 33 H4  24H11QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMG VSWIRQPPGKALEWLAHIFSNDEKSYSTSLKNRLTISKDTSKSQVVLIMTNMDPVDTATYYCARSVVTGG YYYDGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH QDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 34 H5  18G1EVQLLESGGGLVQPGGSLRLSCAASRFTFSTYAMS WVRQAPGKGLEWVSGISGSGVSTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSLIVVI VYALDHWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW LNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 35 H6  17D8EVQLLESGGGLVQPGGYLRLSCAASGFTFSTYAMS WVRQAPGKGLEWVSAISGSGVSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSLIVV MVYVLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQ DWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 36 H7  26H11EVQLLESGGGLVQPGGYLRLSCAASGFTFSTYAMS WVRQAPGKGLEWVSAISGSGVSTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSLIVV MVYVLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQ DWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 37 H8  12E4EVQLLESGGGLVQPGGSLRLSCAASRFTFSTYAMS 12C11WVRQAPGKGLEWVSGISGSGVSTYYADSVKGRFT ISRDNSKNTLYLQMNSLRAEDTAVYYCAKSLIVVIVYALDYWGQGTLVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHK PSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK 38 H9 21H2 QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAGKGLEWIGRIYTSGSTNYNPSLKSRVTMS KDTSKNQFSLKLRSVTAADTAVYYCARDPDGDYYYYGMDVWGQGTSVTVSSASTKGPSVFPLAPCSRS TSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDH KPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 39H10 21B4 QVQLQESGPGLVKPSETLSLTCTVSGGSISSYFWSWIRQPAGKGLEWIGRIYTSGSTNYNPSLKSRVTMSI DTSKNQFSLKLSSVTAADTAVYYCARDPDGDYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTS ESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKP SNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK 40 H1118B11.1 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDAWM 18B11.2SWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVK GRFTISRDDSKNTLYLQMNSLKTEDTAVYFCTSTYSSGWYVWDYYGMDVWGQGTTVTVSSASTKGPSV FPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT QTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISK TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 41 H12 20D4 QVQLVQSGAEVKKPGASVKVSCKVSGYTLTDLSMHWVRQAPGKGLEWMGGFDPEDGETIYAQKFQGRI TMTEDTSTDTAYMELSSLRSEDTAVYYCASIVVVPAAIQSYYYYYGMGVWGQGTTVTVSSASTKGPSVF PLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQT YTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKT KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 H13 46D11 QVTLKEAGPVLVKPTETLTLTCTVSGFSLSNARMGVNWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTI SKDTSKSQVVLTMTNMDPVDTATYYCARVRIAGDYYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPC SRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCN VDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK43 H14 39F11 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGIHWVRQAPGKGLEWVAVIWYDGSDKYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRAAAGLHYYYGMDVWGQGTTVTVSSASTKGPSVFP LAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTY TCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 44 H15 39F7 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGIHWVRQAPGKGLEWVAVIWYDGSIKYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCARDRAAAGLHYYYGMDVWGQGTTVTVSSASTKGPSVFPL APCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYT CNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV QFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 45 H16 39G5 QVQLVESGGGVVQPGRSLRLSCAVSGFTFSSYGIHWVRQAPGKGLEWVAVIWYDGSDKYYGDSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRAAAGLHYYYGMDVWGQGTTVTVSSASTKGPSVFP LAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTY TCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 46 H17 40D2 QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYNWSWIRQHPGKGLEWIGNIYYSGSTYYNPSLKSRVT ISVDTSKNQFSLKLRSVTAADTAVYYCARENIVVIPAAIFAGWFDPWGQGTLVTVSSASTKGPSVFPLAPC SRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCN VDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK47 H18 37D3 EVHLVESGGGLAKPGGSLRLSCAASGFTFRNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVK GRFTISRDDSKNTLYLQMNSLKTEDTAEYYCITDRVLSYYAMAVWGQGTTVTVSSASTKGPSVFPLAPC SRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCN VDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Again, each of the exemplary heavy chains (H1, H2, H3 etc.) listed inTable 1B and 6A, infra, can be combined with any of the exemplary lightchains shown in Table 1A and 6A, infra, to form an antibody. Examples ofsuch combinations include H1 combined with any of L1 through L18; H2combined with any of L1 through L18; H3 combined with any of L1 throughL18, and so on. In some instances, the antibodies include at least oneheavy chain and one light chain from those listed in Tables 1A and 1Band 6A, infra; particular examples pairings of light chains and heavychains include L1 with H1, L2 with H2, L3 with H3, L4 with H4, L5 withH5, L6 with H6, L7 with H7, L8 with H8, L9 with H9, L10 with H10, L11with H11, L12 with H12, L13 with H13, L14 with H14, L15 with H15, L16with H16, L17 with H17, and L18 with H18. In addition to antigen bindingproteins comprising a heavy and a light chain from the same clone, aheavy chain from a first clone can be paired with a light chain from asecond clone (e.g., a heavy chain from 46D11 paired with a light chainfrom 16H7 or a heavy chain from 16H7 paired with a light chain from46D11). Generally, such pairings can include VL with 90% or greaterhomology can be paired with the heavy chain of the naturally occurringclone. In some instances, the antibodies comprise two different heavychains and two different light chains listed in Tables 1A and 1B and 6A,infra. In other instances, the antibodies contain two identical lightchains and two identical heavy chains. As an example, an antibody orimmunologically functional fragment can include two H1 heavy chains andtwo L1 light chains, or two H2 heavy chains and two L2 light chains, ortwo H3 heavy chains and two L3 light chains and other similarcombinations of pairs of light chains and pairs of heavy chains aslisted in Tables 1A and 1B and 6A, infra.

In another aspect of the instant disclosure, “hemibodies” are provided.A hemibody is a monovalent antigen binding protein comprising (i) anintact light chain, and (ii) a heavy chain fused to an Fc region (e.g.,an IgG2 Fc region of SEQ ID NO:441), optionally via a linker, The linkercan be a (G₄S)_(x) linker where “x” is a non-zero integer (e.g., (G₄S)₈;SEQ ID NO:440). Hemibodies can be constructed using the provided heavyand light chain components. Specific examples of hemibodies aredisclosed in Example 14.

Other antigen binding proteins that are provided are variants ofantibodies formed by combination of the heavy and light chains shown inTables 1A and 1B and 6A, infra and comprise light and/or heavy chainsthat each have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% identity to the amino acid sequences of these chains. In someinstances, such antibodies include at least one heavy chain and onelight chain, whereas in other instances the variant forms contain twoidentical light chains and two identical heavy chains.

Variable Domains of Antigen Binding Proteins

Also provided are antigen binding proteins that contain an antibodyheavy chain variable region selected from the group consisting ofV_(H)1, V_(H)2, V_(H)3, V_(H)4, V_(H)5, V_(H)6, V_(H)7, V_(H)8, V_(H)9,V_(H)10, V_(H)11, V_(H)12, V_(H)13, V_(H)14, V_(H)15, V_(H)16, V_(H)17and V_(H)18 as shown in Table 2B and/or an antibody light chain variableregion selected from the group consisting of V_(L)1, V_(L)2, V_(L)3,V_(L)4, V_(L)5, V_(L)6, V_(L)7, V_(L)8, V_(L)9, V_(L)10, V_(L)11,V_(L)12, V_(L)13, V_(L)14, V_(L)15, V_(L)16, V_(L)17 and V_(L)18 asshown in Table 2A, and immunologically functional fragments,derivatives, muteins and variants of these light chain and heavy chainvariable regions.

TABLE 2A Exemplary Antibody Variable Light (V_(L)) Chains Contained SEQID in Clone Designation NO. Amino Acid Sequence 17C3 V_(L)1 48SYVLTQPPSVSVAPGQTARITCGGNNIGSQSVHWY QQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTK LTVL 22H5 V_(L)2 49SYVLTQPPSVSVAPGQTARITCGGNNIGSQSVHWY QQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDNTSDHVVFGGGTK LTVL 16H7 V_(L)3 50SYVLTQPPSVSVAPGQTARITCGGNNIGSESVHWY 24H11QQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTA TLTISRVEAGDEADYYCQVWDGNSDHVVFGGGTKLTVL 18G1 V_(L)4 51 EIVLTQSPGTLSLSPGERATLSCRASQNFDSSYLAWYQQKPGQAPRLLIYGTSSRATGIPDRFSGIGSGTDFTLTINRLEPEDFAMYYCQQYGGSPLTFGGGTEVEIK 17D8 V_(L)5 52EIVLTQSPGTLSLSPGERATLSCRASQSVSGNYLA WYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSAPLTFGGGTKV EIK 26H11 V_(L)6 53EIVLTQSPGTLSLSPGERATLSCRASQSVSGNYLA WYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAMYYCQQYGSSPLTFGGGSKV EIK 12E4 V_(L)7 54EIVLTQSPGTLSLSPGERATLSCRASQNFDSNYLA WYQQKPGQAPRLLIYGASSRATGIPDNFSGSGSGTDFTLTISRLEPEDFAMYYCQQYGSSPLTFGGGTKV EIK 12C11 V_(L)8 55EIVLTQSPGTLSLSPGERATLSCRASQNFDSSSLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAMYYCQQCGSSPLTFGGGTKVEIK 21H2 V_(L)9 56EIVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAW 21B4HQQKPGQGLRLLIYGASSRATGIPDRFSGSGSGTDF TLTISRLEPEDFAVYYCQQYGSSFTFGGGTRVEIK18B11.1 V_(L)10 57 DIVMTQSPLSLPVTPGEPASISCRSSQSLLYYNGFTYLDWFLQKPGQSPHLLIYLGSNRASGVPDRFSGSV SGTDFTLKISRVEAEDVGVYYCMQSLQTPFTFGPGTKVDIK 18B11.2 V_(L)11 58 EIVMTQSPATLSVSPGERATLSCRASQSVNSNLAWYQQKPGQAPRLLIYGVSTRATGIPARFSGSGSGTEFTLTIRSLQSEDFAVYYCQQYNNWPPTFGQGTKVEIK 20D4 V_(L)12 59DIQLTQSPSSLSASIGDRVTITCRASQDIRYDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT LTVSSLQPEDFATYYCLQHNSYPLTFGGGTKVEIE46D11 V_(L)13 60 DIQMTQSPSSVSASVGDRVTITCRASQGISIWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANDFPITFGQGTRLEIK 40D2 V_(L)14 61DFVMTQTPLSLSVTPGQPASISCKSSQSLLQSDGKT YLYWYLQKPGQPPHLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSIQLPRTFGQG TKVEIK 37D3 V_(L)15 62DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYN FLDWYLQKPGQSPQLLIYLGSDRASGVPDRFSGSGSGTEFTLKISRVEAEDVGLYYCMQALQTPCSFGQG TKLEIK 39F7 V_(L)16 63EIVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSGSSPLTFGGGTEVEIK 39F11 V_(L)17 64EIVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQKPGQAPSLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSGSSPLTFGGGTKVEIK 39G5 V_(L)18 65EIVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQKPGQAPRLLIYGASFRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSGSSPLTFGGGTKVEIK

TABLE 2B Exemplary Antibody Variable Heavy (V_(H)) Chains Contained SEQID in Clone Designation NO. Amino Acid Sequence 17C3 V_(H)1 66QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMG VSWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARILLLGA YYYYGMDVWGQGTTVTVSS 22H5 V_(H)2 67QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMG VSWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARILLVGA YYYCGMDVWGQGTTVTVSS 16H7 V_(H)3 68QVTLKESGPVLVKPTETLTLTCTVSGFSLNNARMG VSWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKSQVVLIMTNMDPVDTATYYCARSVVTGG YYYDGMDVWGQGTTVTVSS 24H11 V_(H)4 69QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMG VSWIRQPPGKALEWLAHIFSNDEKSYSTSLKNRLTISKDTSKSQVVLIMTNMDPVDTATYYCARSVVTGG YYYDGMDVWGQGTTVTVSS 18G1 V_(H)5 70EVQLLESGGGLVQPGGSLRLSCAASRFTFSTYAMS WVRQAPGKGLEWVSGISGSGVSTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSLIVVI VYALDHWGQGTLVTVSS 17D8 V_(H)6 71EVQLLESGGGLVQPGGYLRLSCAASGFTFSTYAMS WVRQAPGKGLEWVSAISGSGVSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSLIVV MVYVLDYWGQGTLVTVSS 26H11 V_(H)7 72EVQLLESGGGLVQPGGYLRLSCAASGFTFSTYAMS WVRQAPGKGLEWVSAISGSGVSTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSLIVV MVYVLDYWGQGTLVTVSS 12E4 V_(H)8 73EVQLLESGGGLVQPGGSLRLSCAASRFTFSTYAMS 12C11WVRQAPGKGLEWVSGISGSGVSTYYADSVKGRFT ISRDNSKNTLYLQMNSLRAEDTAVYYCAKSLIVVIVYALDYWGQGTLVTVSS 21H2 V_(H)9 74 QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAGKGLEWIGRIYTSGSTNYNPSLKSRVTMS KDTSKNQFSLKLRSVTAADTAVYYCARDPDGDYYYYGMDVWGQGTSVTVSS 21B4 V_(H)10 75 QVQLQESGPGLVKPSETLSLTCTVSGGSISSYFWSWIRQPAGKGLEWIGRIYTSGSTNYNPSLKSRVTMS IDTSKNQFSLKLSSVTAADTAVYYCARDPDGDYYYYGMDVWGQGTTVTVSS 18B11.1 V_(H)11 76 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVK GRFTISRDDSKNTLYLQMNSLKTEDTAVYFCTSTYSSGWYVWDYYGMDVWGQGTTVTVSS 18B11.2 V_(H)11 77EVQLVESGGGLVKPGGSLRLSCAASGFTFSDAWM SWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYFCTSTY SSGWYVWDYYGMDVWGQGTTVTVSS 20D4V_(H)12 78 QVQLVQSGAEVKKPGASVKVSCKVSGYTLTDLSMHWVRQAPGKGLEWMGGFDPEDGETIYAQKFQGR ITMTEDTSTDTAYMELSSLRSEDTAVYYCASIVVVPAAIQSYYYYYGMGVWGQGTTVTVSS 46D11 V_(H)13 79QVTLKEAGPVLVKPTETLTLTCTVSGFSLSNARMG VNWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARVRIAGD YYYYYGMDVWGQGTTVTVSS 40D2 V_(H)14 80QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYN WSWIRQHPGKGLEWIGNIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLRSVTAADTAVYYCARENIVVIP AAIFAGWFDPWGQGTLVTVSS 37D3 V_(H)1581 EVHLVESGGGLAKPGGSLRLSCAASGFTFRNAWM SWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAEYYCITDR VLSYYAMAVWGQGTTVTVSS 39F7 V_(H)16 82QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGIH WVRQAPGKGLEWVAVIWYDGSIKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRAA AGLHYYYGMDVWGQGTTVTVSS 39F11 V_(H)1783 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGIH WVRQAPGKGLEWVAVIWYDGSDKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRA AAGLHYYYGMDVWGQGTTVTVSS 39G5 V_(H)1884 QVQLVESGGGVVQPGRSLRLSCAVSGFTFSSYGIH WVRQAPGKGLEWVAVIWYDGSDKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRA AAGLHYYYGMDVWGQGTTVTVSS

TABLE 2C Coding Sequence for Antibody Variable Light (V_(L)) ChainsContained SEQ ID in Clone Designation NO. Coding Sequence 17C3 V_(L)1 85TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAG TGGCCCCAGGTCAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTCAGAGTGTGCACT GGTACCAGCAGAAGCCAGGCCAGGCCCCTGTCCTGGTCGTCTATGATGATAGCGACCGGCCCTCAG GGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGA AGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGTAGTAGTGATCATGTGGTATTCGG CGGAGGGACCAAGCTGACCGTCCTA 22H5 V_(L)286 TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAG TGGCCCCAGGACAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTCAAAGTGTGCACT GGTACCAGCAGAAGCCAGGCCAGGCCCCTGTCCTGGTCGTCTATGATGATAGCGACCGGCCCTCAG GGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGA AGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAATACTAGTGATCATGTGGTATTCGG CGGGGGGACCAAACTGACCGTCCTA 16H7 V_(L)387 TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAG 24H11TGGCCCCAGGACAGACGGCCAGGATTACCTGTG GGGGAAACAACATTGGAAGTGAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGC TGGTCGTCTATGATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGG GAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGT GTGGGATGGTAATAGTGATCATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA 18G1 V_(L)A 88GAAATTGTGTTGACGCAGTCTCCAGGCACCCTG TCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAATTTTGACAGCAGTTACT TAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCCGGCTCCTCATCTATGGTACATCCAGCAGGG CCACTGGCATCCCAGACAGGTTCAGTGGCATTGGGTCTGGGACAGACTTCACTCTCACCATCAACA GACTGGAGCCTGAAGATTTTGCAATGTATTACTGTCAGCAGTATGGTGGCTCACCGCTCACTTTCGG CGGAGGGACCGAGGTGGAAATCAAA 17D8 V_(L)589 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTG TCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCGGCAACTACT TGGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGG CCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCA GACTGGAGCCTGAAGATTTTGCAGTGTATTATTGTCAGCAGTATGGTAGCGCACCGCTCACTTTCGG CGGAGGGACCAAGGTGGAAATCAAA 26H11 V_(L)690 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTG TCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCGGCAACTACT TGGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGG CCACTGGCATCCCAGACAGATTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCA GACTGGAGCCTGAAGATTTTGCAATGTATTATTGTCAGCAGTATGGTAGCTCACCGCTCACTTTCGGC GGAGGGTCCAAGGTGGAGATCAAA 12E4 V_(L)791 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTG TCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAATTTCGACAGCAACTACT TAGCCTGGTACCAGCAGAAGCCTGGCCAGGCTCCCCGGCTCCTCATCTATGGTGCATCCAGCAGGG CCACTGGCATCCCAGACAACTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCA GACTGGAGCCTGAAGATTTTGCAATGTATTACTGTCAGCAGTATGGTAGTTCACCGCTCACTTTCGG CGGAGGGACCAAGGTGGAAATCAAA 12C11V_(L)8 92 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGGGCCACCCTCTCCT GCAGGGCCAGTCAGAATTTTGACAGCAGCTCCTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTC CCCGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTG GGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAATGTATTACT GTCAGCAGTGTGGTAGCTCACCGCTCACTTTCGGCGGAGGGACCAAGGTGGAAATCAAA 21H2 V_(L)9 93GAAATTGTGTTGACGCAGTCTCCAGGCACCCTG 21B4TCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCT GCAGGGCCAGTCAGAGTGTTAGCAGTACCTACTTAGCCTGGCACCAGCAGAAACCTGGCCAGGGTC TTAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGG GTCTGGGACAGACTTCACTCTTACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGT CAGCAGTATGGAAGCTCATTCACTTTCGGCGGAGGGACCAGGGTGGAGATCAAA 18B11.1 V_(L)10 94GATATTGTGATGACTCAGTCTCCACTCTCCCTGC CCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGTATTATAATGG ATTCACCTATTTGGATTGGTTCCTGCAGAAGCCAGGGCAGTCTCCACATCTCCTGATCTATTTGGGTT CTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGTTTCAGGCACAGATTTTACACTGA AAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTATTGCATGCAGTCTCTGCAAACTCCATT CACTTTCGGCCCTGGGACCAAAGTGGATATCAAA18B11.2 V_(L)11 95 GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCT GCAGGGCCAGTCAGAGTGTTAACAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCA GGCTCCTCATTTATGGTGTATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTC TGGGACAGAGTTCACTCTCACCATCCGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAG CAGTATAATAACTGGCCTCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA 20D4 V_(L)12 96 GACATACAGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTATAGGAGACAGAGTCACCATCACTT GCCGGGCAAGTCAGGACATTAGATATGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTA AGCGCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCTTCAAGGTTCAGCGGCAGTGGAT CTGGGACAGAATTCACTCTCACAGTCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCT ACAGCATAATAGTTACCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCGAA 46D11 V_(L)13 97GACATCCAGATGACCCAGTCTCCCTCTTCCGTGT CTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCATCTGGTTAG CCTGGTATCAGCAGAAACCTGGGAAAGCCCCTAAACTCCTGATCTATGCTGCATCCAGTTTGCAAAG TGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCT GCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACGATTTCCCGATCACCTTCGGCCAA GGGACACGACTGGAGATTAAA 40D2 V_(L)14 98GATTTTGTGATGACCCAGACTCCACTCTCTCTGT CCGTCACCCCTGGACAGCCGGCCTCCATCTCCTGCAAGTCTAGTCAGAGCCTCCTACAGAGTGATGG AAAGACCTATTTGTATTGGTACCTGCAGAAGCCAGGCCAGCCTCCACATCTCCTGATCTATGAAGTT TCCAACCGATTCTCTGGAGTGCCAGATAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACACTG AAAATCAGCCGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAAGTATACAGCTTCCTC GGACGTTCGGCCAAGGGACCAAGGTGGAAATCA AA37D3 V_(L)15 99 GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTG CAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTTTTTGGATTGGTACCTACAGAAGCC AGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTGATCGGGCCTCCGGGGTCCCTGACAGGTTC AGTGGCAGTGGATCAGGCACAGAGTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGG CTTTATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCA AA 39F7 V_(L)16 100GAAATTGTGTTGACGCAGTCTCCAGGCACCCTG TCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGTAGCACCTATT TAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGG CCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCA GACTGGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTCTGGTAGCTCACCGCTCACTTTCGGC GGAGGGACCGAGGTGGAGATCAAA 39F11V_(L)17 101 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCT GCAGGGCCAGTCAGAGTGTTAGCAGCACCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTC CCAGTCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGG GTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAGGATTTTGCAGTGTATTACTGT CAGCAGTCTGGTAGCTCACCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA 30G5 V_(L)18 102GAAATTGTGTTGACGCAGTCTCCAGGCACCCTG TCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCACCTACT TAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCTTCAGGGC CACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAG ACTGGAGCCTGAGGATTTTGCAGTGTATTACTGTCAGCAGTCTGGTAGCTCACCTCTCACTTTCGGCG GAGGGACCAAGGTGGAGATCAAA

TABLE 2D Coding Sequence for Antibody Variable Heavy (V_(H)) ChainsContained SEQ ID in Clone Designation NO. Coding Sequence 17C3 V_(H)1103 CAGGTCACCTTGAAGGAGTCTGGTCCTGTGCTG GTGAAACCCACAGAGACCCTCACGCTGACCTGCACCGTCTCTGGGTTCTCACTCAGCAATGCTAGAA TGGGTGTGAGCTGGATCCGTCAGCCCCCAGGGAAGGCCCTGGAGTGGCTTGCACACATTTTTTCGAA TGACGAAAAATCCTACAGCACATCTCTGAAGAGCAGGCTCACCATCTCCAAGGACACCTCCAAAAG CCAGGTGGTCCTTACCATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACGGAT ATTATTACTGGGAGCTTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTC TCCTCA 22H5 V_(H)2 104CAGGTCACCTTGAAGGAGTCTGGTCCTGTGCTG GTGAAACCCACAGAGACCCTCACGCTGACCTGCACCGTCTCTGGGTTCTCACTCAGCAATGCTAGAA TGGGTGTGAGCTGGATCCGTCAGCCCCCAGGGAAGGCCCTGGAGTGGCTTGCACACATTTTTTCGAA TGACGAAAAATCCTACAGCACATCTCTGAAGAGCAGGCTCACCATCTCCAAGGACACCTCCAAAAG CCAGGTGGTCCTTACCATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACGGAT ATTATTAGTGGGAGCTTACTACTACTGCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTC TCCTCA 16H7 V_(H)3 105CAGGTCACCTTGAAGGAGTCTGGTCCTGTGCTG GTGAAACCCACAGAGACCCTCACGCTGACCTGCACCGTCTCTGGGTTCTCACTCAACAATGCTAGAA TGGGTGTGAGCTGGATCCGTCAGCCCCCAGGGAAGGCCCTGGAGTGGCTTGCACACATTTTTTCGAA TGACGAAAAATCCTACAGCACATCTCTGAAGAGCAGGCTCACCATCTCCAAGGACACCTCCAAAAG CCAGGTGGTCCTAATTATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACGGTC AGTAGTAACTGGCGGCTACTACTACGACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGT CTCCTCA 24H11 V_(H)4 106CAGGTCACCTTGAAGGAGTCTGGTCCTGTGCTG GTGAAACCCACAGAGACCCTCACGCTGACCTGCACCGTCTCTGGGTTCTCACTCAGCAATGCTAGAA TGGGTGTGAGCTGGATCCGTCAGCCCCCAGGGAAGGCCCTGGAGTGGCTTGCACACATTTTTTCGAA TGACGAAAAATCCTACAGCACATCTCTGAAGAACAGGCTCACCATCTCCAAGGACACCTCCAAAAG CCAGGTGGTCCTTATTATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACGGTCA GTAGTGACTGGCGGCTACTACTACGACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTC TCCTCA 18G1 V_(H)5 107GAGGTGCAGCTGTTGGAGTCTGGGGGAGGGTTG GTACAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTAGATTCACCTTTAGCACCTATGCCA TGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTAGTGGTAGTGGTG TCAGCACACACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACA CGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCCC TCATTGTAGTAATAGTATATGCCCTTGACCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 17D8 V_(H)6 108GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTG GTACAGCCGGGGGGGTACCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGTACCTATGCCA TGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGGTCTCAGCTATCAGTGGTAGTGGTG TTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACA CGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCCC TTATTGTAGTAATGGTGTATGTCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 26H11 V_(H)7 109GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTG GTACAGCCGGGGGGGTACCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGCACCTATGCCA TGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGGTCTCAGCTATTAGTGGCAGTGGTG TGAGCACAAACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACA CGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCCC TTATTGTAGTAATGGTGTATGTCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 12E4 V_(H)8 110GAGGTGCAGCTGTTGGAGTCTGGGGGAGGGTTG 12C11GTACAGCCGGGGGGGTCCCTGAGACTCTCCTGT GCAGCCTCTAGATTCACCTTTAGCACCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGC TGGAGTGGGTCTCAGGTATTAGTGGTAGTGGTGTTAGCACATACTACGCAGACTCCGTGAAGGGCC GGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCG AGGACACGGCCGTATATTACTGTGCGAAATCCCTTATTGTAGTAATAGTATATGCCCTTGACTACTG GGGCCAGGGAACCCTGGTCACCGTCTCCTCA 21H2V_(H)9 111 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGC ACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCGCCGGGAAGGGA CTGGAGTGGATTGGGCGTATCTATACCAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGG GTCACCATGTCAAAAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGGTCTGTGACCGCCGCG GACACGGCCGTGTATTACTGTGCGAGAGATCCGGACGGTGACTACTACTACTACGGTATGGACGTC TGGGGCCAAGGGACCTCGGTCACCGTCTCCTCA 21B4V_(H)10 112 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGC ACTGTCTCTGGTGGCTCCATCAGTAGTTACTTCTGGAGCTGGATCCGGCAGCCCGCCGGGAAGGGA CTGGAGTGGATTGGGCGTATCTATACCAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGA GTCACCATGTCAATAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCGG ACACGGCCGTGTATTACTGTGCGAGAGATCCGGACGGTGACTACTACTACTACGGTATGGACGTCT GGGGCCAAGGGACCACGGTCACCGTCTCCTCA18B11.1 V_(H)11 113 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTG 18B11.2GTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGT GCAGCCTCTGGATTCACTTTCAGTGACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGC TGGAGTGGGTTGGCCGTATTAAAAGCAAAACTGATGGTGGGACAACAGACTACGCTGCACCCGTGA AAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACTCTGTATCTGCAAATGAACAGCCTGA AAACCGAGGACACAGCCGTGTATTTTTGTACCTCTACGTATAGCAGTGGCTGGTACGTATGGGACT ACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 20D4 V_(H)12 114 CAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGC AAGGTTTCGGGATACACCCTCACTGATTTATCCATGCACTGGGTGCGACAGGCTCCTGGAAAAGGGC TTGAGTGGATGGGAGGTTTTGATCCTGAAGATGGTGAAACAATCTACGCACAGAAGTTCCAGGGCA GAATCACCATGACCGAGGACACATCTACAGACACAGCCTACATGGAGCTGAGCAGCCTGAGATCTG AGGACACGGCCGTGTATTACTGTGCAAGTATTGTAGTAGTCCCAGCTGCTATACAGAGTTACTACTA CTACTACGGTATGGGCGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCC 46D11 V_(H)13 115 CAGGTCACCTTGAAGGAGGCTGGTCCTGTGTTGGTGAAACCCACAGAGACCCTCACGTTGACCTGC ACCGTCTCTGGGTTCTCACTCAGCAATGCTAGAATGGGTGTGAACTGGATCCGTCAGCCCCCAGGGA AGGCCCTGGAGTGGCTTGCACACATTTTTTCGAATGACGAAAAATCCTACAGCACATCTCTGAAGAG CAGGCTCACCATCTCCAAGGACACCTCCAAAAGCCAGGTGGTCCTTACCATGACCAACATGGACCC TGTGGACACAGCCACATATTACTGTGCACGGGTTCGTATAGCAGGTGATTACTACTACTACTACGGT ATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 40D2 V_(H)14 116 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCA CTGTCTCTGGTGGCTCCATCAGCAGTGGTGGTTACAACTGGAGCTGGATCCGCCAGCACCCAGGGAA GGGCCTGGAGTGGATTGGGAACATCTATTACAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAG TCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGATCTGTGACTGCC GCGGACACGGCCGTGTATTACTGTGCGAGAGAGAATATTGTAGTAATACCAGCTGCTATATTCGCGG GTTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 37D3 V_(H)15 117 GAGGTGCACCTGGTGGAGTCTGGGGGAGGCTTGGCAAAGCCTGGGGGGTCCCTTAGACTCTCCTGT GCAGCCTCTGGATTCACTTTCAGAAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGG CTGGAATGGGTTGGCCGTATTAAAAGCAAAACTGATGGTGGGACAACAGACTACGCTGCACCCGTG AAAGGCAGATTCACCATCTCGAGAGATGATTCAAAAAACACGCTGTATCTGCAAATGAACAGCCTG AAAACCGAGGACACAGCCGAGTATTACTGTATCACAGATCGGGTGCTAAGCTACTACGCTATGGCC GTCTGGGGCCAAGGGACCACGGTCACCGTCTCC TCA39F7 V_(H)16 118 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGT GCAGCGTCTGGATTCACCTTCAGTAACTATGGCATTCACTGGGTCCGCCAGGCTCCAGGCAAGGGG CTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTATTAAATACTATGCAGACTCCGTGAAGGGC CGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCC GAGGACACGGCTGTGTATTACTGTGCGAGAGATAGGGCAGCAGCTGGTCTCCACTACTACTACGGT ATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 39F11 V_(H)17 119 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGT GCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATCCACTGGGTCCGCCAGGCTCCAGGCAAGGGG CTGGAATGGGTGGCAGTTATATGGTATGATGGAAGTGATAAATACTATGCAGACTCCGTGAAGGGC CGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTACAAATGAACAGCCTGAGAGCC GAGGACACGGCTGTGTATTACTGTGCGAGAGATAGGGCAGCAGCTGGTCTCCACTATTATTACGGT ATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 39G5 V_(H)18 120 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGT GCAGTGTCTGGATTCACCTTCAGTAGCTATGGCATCCACTGGGTCCGCCAGGCTCCAGGCAAGGGGC TGGAATGGGTGGCAGTTATATGGTATGATGGAAGTGATAAATACTATGGAGACTCCGTGAAGGGCC GATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTACAAATGAACAGCCTGAGAGCCG AGGACACGGCTGTGTATTACTGTGCGAGAGATAGGGCAGCAGCTGGTCTCCACTATTATTACGGTAT GGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

Each of the heavy chain variable regions listed in Table 2B can becombined with any of the light chain variable regions shown in Table 2Ato form an antigen binding protein. Examples of such combinationsinclude V_(H)1 combined with any of V_(L)1, V_(L)2, V_(L)3, V_(L)4,V_(L)5, V_(L)6, V_(L)7, V_(L)8, V_(L)9, V_(L)10, V_(L)11, V_(L)12,V_(L)13, V_(L)14, V_(L)15, V_(L)16, V_(L)17 or V_(L)18; V_(H)2 combinedwith any of V_(L)1, V_(L)2, V_(L)3, V_(L)4, V_(L)5, V_(L)6, V_(L)7,V_(L)8, V_(L)9, V_(L)10, V_(L)11, V_(L)12, V_(L)13, V_(L)14, V_(L)15,V_(L)16, V_(L)17 or V_(L)18; V_(H)3 combined with any of V_(L)1, V_(L)2,V_(L)3, V_(L)4, V_(L)5, V_(L)6, V_(L)7, V_(L)8, V_(L)9, V_(L)10,V_(L)11, V_(L)12, V_(L)13, V_(L)14, V_(L)15, V_(L)16, V_(L)17 orV_(L)18; and so on.

In some instances, the antigen binding protein includes at least oneheavy chain variable region and/or one light chain variable region fromthose listed in Tables 2A and 2B. In some instances, the antigen bindingprotein includes at least two different heavy chain variable regionsand/or light chain variable regions from those listed in Table 2B. Anexample of such an antigen binding protein comprises (a) one V_(H)1, and(b) one of V_(H)2, V_(H)3, V_(H)4, V_(H)5, V_(H)6, V_(H)7, V_(H)8,V_(H)9, V_(H)10, V_(H)11, V_(H)12, VH₁₃, VH₁₄, VH₁₅, VH₁₆, VH₁₇ or VH₁₈.Another example comprises (a) one V_(H)2, and (b) one of V_(H)1, V_(H)3,V_(H)4, V_(H)5, V_(H)6, V_(H)7, V_(H)8, V_(H)9, V_(H)10, V_(H)11,V_(H)12, V_(H)13, V_(H)14, V_(H)15, V_(H)16, V_(H)17 or V_(H)18. Againanother example comprises (a) one V_(H)3, and (b) one of V_(H)1, V_(H)2,V_(H)4, V_(H)5, V_(H)6, V_(H)7, V_(H)8, V_(H)9, V_(H)10, V_(H)11,V_(H)12, V_(H)13, V_(H)14, V_(H)15 V_(H)16, V_(H)17 or V_(H)18, etc.

Again another example of such an antigen binding protein comprises (a)one V_(L)1, and (b) one of V_(L)2, V_(L)3, V_(L)4, V_(L)5, V_(L)6,V_(L)7, V_(L)8, V_(L)9, V_(L)10, V_(L)11, V_(L)12, V_(L)13, V_(L)14,V_(L)15, V_(L)16, V_(L)17, or V_(L)18. Again another example of such anantigen binding protein comprises (a) one V_(L)2, and (b) one of V_(L)1,V_(L)3, V_(L)4, V_(L)5, V_(L)6, V_(L)7, V_(L)8, V_(L)9, V_(L)10, V_(L)11or V_(L)12. Again another example of such an antigen binding proteincomprises (a) one V_(L)3, and (b) one of V_(L)1, V_(L)2, V_(L)4, V_(L)5,V_(L)6, V_(L)7, V_(L)8, V_(L)9, V_(L)10, V_(L)11, V_(L)12, V_(L)13,V_(L)14, V_(L)15, V_(L)16, V_(L)17, or V_(L)18, etc.

The various combinations of heavy chain variable regions can be combinedwith any of the various combinations of light chain variable regions.

In other embodiments, an antigen binding protein comprises two identicallight chain variable regions and/or two identical heavy chain variableregions. As an example, the antigen binding protein can be an antibodyor immunologically functional fragment thereof that includes two lightchain variable regions and two heavy chain variable regions incombinations of pairs of light chain variable regions and pairs of heavychain variable regions as listed in Tables 2A and 2B.

Some antigen binding proteins that are provided comprise a heavy chainvariable domain comprising a sequence of amino acids that differs fromthe sequence of a heavy chain variable domain selected from V_(H)1,V_(H)2, V_(H)3, V_(H)4, V_(H)5, V_(H)6, V_(H)7, V_(H)8, V_(H)9, V_(H)10,V_(H)11, V_(H)12, V_(H)13, V_(H)14, V_(H)15, V_(H)16, V_(H)17 andV_(H)18 at only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15amino acid residues, wherein each such sequence difference isindependently either a deletion, insertion or substitution of one aminoacid, with the deletions, insertions and/or substitutions resulting inno more than 15 amino acid changes relative to the foregoing variabledomain sequences. The heavy chain variable region in some antigenbinding proteins comprises a sequence of amino acids that has at least70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% sequence identity to the aminoacid sequences of the heavy chain variable region of V_(H)1, V_(H)2,V_(H)3, V_(H)4, V_(H)5, V_(H)6, V_(H)7, V_(H)8, V_(H)9, V_(H)10,V_(H)11, V_(H)12, V_(H)13, V_(H)14, V_(H)15, V_(H)16, V_(H)17 andV_(H)18.

Certain antigen binding proteins comprise a light chain variable domaincomprising a sequence of amino acids that differs from the sequence of alight chain variable domain selected from V_(L)1, V_(L)2, V_(L)3,V_(L)4, V_(L)5, V_(L)6, V_(L)7, V_(L)8, V_(L)9, V_(L)10, V_(L)11,V_(L)12, V_(L)13, V_(L)14, V_(L)15, V_(L)16, V_(L)17 and V_(L)18 at only1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues,wherein each such sequence difference is independently either adeletion, insertion or substitution of one amino acid, with thedeletions, insertions and/or substitutions resulting in no more than 15amino acid changes relative to the foregoing variable domain sequences.The light chain variable region in some antigen binding proteinscomprises a sequence of amino acids that has at least 70%, 75%, 80%,85%, 90%, 95%, 97% or 99% sequence identity to the amino acid sequencesof the light chain variable region of V_(L)1, V_(L)2, V_(L)3, V_(L)4,V_(L)5, V_(L)6, V_(L)7, V_(L)8, V_(L)9, V_(L)10, V_(L)11, V_(L)12,V_(L)13, V_(L)14, V_(L)15, V_(L)16, V_(L)17 or V_(L)18.

In additional instances, antigen binding proteins comprise the followingpairings of light chain and heavy chain variable domains: V_(L)1 withV_(H)1, V_(L)2 with V_(H)2, V_(L)2 with V_(H)3, V_(L)3 with V_(H)4,V_(L)4 with V_(H)5, V_(L)5 with V_(H)6, V_(L)6 with V_(H)7, V_(L)7 withV_(H)8, V_(L)8 with V_(H)8, V_(L)9 with V_(H)9, V_(L)9 with V_(H)10,V_(L)10 with V_(H)11, V_(L)11 with V_(H)11, V_(L)12 with V_(H)12,V_(L)13 with V_(H)13, V_(L)14 with V_(H)14, V_(L)15 with V_(H)15,V_(L)16 with V_(H)16, V_(L)17 with V_(H)17 and V_(L)18 with V_(H)18. Insome instances, the antigen binding proteins in the above pairings cancomprise amino acid sequences that have 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% sequence identity with the specified variabledomains.

Still other antigen binding proteins, e.g., antibodies orimmunologically functional fragments, include variant forms of a variantheavy chain and a variant light chain as just described.

Antigen Binding Protein CDRs

In various embodiments, the antigen binding proteins disclosed hereincan comprise polypeptides into which one or more CDRs are grafted,inserted and/or joined. An antigen binding protein can have 1, 2, 3, 4,5 or 6 CDRs. An antigen binding protein thus can have, for example, oneheavy chain CDR1 (“CDRH1”), and/or one heavy chain CDR2 (“CDRH2”),and/or one heavy chain CDR3 (“CDRH3”), and/or one light chain CDR1(“CDRL1”), and/or one light chain CDR2 (“CDRL2”), and/or one light chainCDR3 (“CDRL3”). Some antigen binding proteins include both a CDRH3 and aCDRL3. Specific heavy and light chain CDRs are identified in Tables 3Aand 3B, respectively and in Table 6C, infra.

Complementarity determining regions (CDRs) and framework regions (FR) ofa given antibody can be identified using the system described by Kabatet al., in Sequences of Proteins of Immunological Interest, 5th Ed., USDept. of Health and Human Services, PHS, NIH, NIH Publication no.91-3242, 1991. As desired, the CDRs can also be redefined according analternative nomenclature scheme, such as that of Chothia (see Chothia &Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature342:878-883 or Honegger & Pluckthun, 2001, J. Mol. Biol. 309:657-670).Certain antibodies that are disclosed herein comprise one or more aminoacid sequences that are identical or have substantial sequence identityto the amino acid sequences of one or more of the CDRs presented inTable 3A (CDRHs) and Table 3B (CDRLs) and Table 6C, infra.

TABLE 3A Exemplary CDRH Sequences SEQ Contained ID in Clone NO:Reference Designation Sequence 20D4 121 V_(H)12 CDRH1-1 DLSMH 17C3 122V_(H)1 CDRH1-2 NARMGVS 22H5 V_(H)2 16H7 V_(H)3 24H11 V_(H)4 18B11.1 123V_(H)11 CDRH1-3 DAWMS 18B11.2 V_(H)11 18G1 124 V_(H)5 CDRH1-4 TYAMS12C11 V_(H)8 12E4 V_(H)8 17D8 V_(H)6 26H11 V_(H)7 21B4 125 V_(H)10CDRH1-5 SYFWS 46D11 126 V_(H)13 CDRH1-6 NARMGVN 37D3 127 V_(H)15 CDRH1-7NAWMS 39F11 128 V_(H)17 CDRH1-8 SYGIH 39G5 V_(H)18 39F7 129 V_(H)16CDRH1-9 NYGIH 40D2 130 V_(H)14 CDRH1-10 SGGYNWS 21H2 131 V_(H)9 CDRH1-11SYYWS 20D4 132 V_(H)12 CDRH2-1 GFDPEDGETIYAQKFQG 17C3 133 V_(H)1 CDRH2-2HIFSNDEKSYSTSLKS 22H5 V_(H)2 16H7 V_(H)3 46D11 V_(H)13 24H11 134 V_(H)4CDRH2-3 HIFSNDEKSYSTSLKN 18B11.1 135 V_(H)11 CDRH2-4 RIKSKTDGGTTDYAAP18B11.2 V_(H)11 VKG 37D3 V_(H)15 18G1 136 V_(H)5 CDRH2-5GISGSGVSTHYADSVKG 12C11 137 V_(H)8 CDRH2-6 GISGSGVSTYYADSVKG 12E4 V_(H)817D8 138 V_(H)6 CDRH2-7 AISGSGVSTYYADSVKG 26H11 139 V_(H)7 CDRH2-8AISGSGVSTNYADSVKG 21B4 140 V_(H)10 CDRH2-9 RIYTSGSTNYNPSLKS 21H2 V_(H)939F11 141 V_(H)17 CDRH2-10 VIWYDGSDKYYADSVKG 39F7 142 V_(H)16 CDRH2-11VIWYDGSIKYYADSVKG 39G5 143 V_(H)18 CDRH2-12 VIWYDGSDKYYGDSVKG 40D2 144V_(H)14 CDRH2-13 NIYYSGSTYYNPSLKS 20D4 145 V_(H)12 CDRH3-1IVVVPAAIQSYYYYY GMGV 17C3 146 V_(H)1 CDRH3-2 ILLLGAYYYYGMDV 22H5 147V_(H)2 CDRH3-3 ILLVGAYYYCGMDV 16H7 148 V_(H)3 CDRH3-4 SVVTGGYYYDGMDV24H11 V_(H)4 18B11.1 149 V_(H)11 CDRH3-5 TYSSGWYVWDYYGMDV 18B11.2V_(H)11 18G1 150 V_(H)5 CDRH3-6 SLIVVIVYALDH 12C11 151 V_(H)8 CDRH3-7SLIVVIVYALDY 12E4 V_(H)8 17D8 152 V_(H)6 CDRH3-8 SLIVVMVYVLDY 26H11V_(H)7 21B4 153 V_(H)10 CDRH3-9 DPDGDYYYYGMDV 21H2 V_(H)9 46D11 154V_(H)13 CDRH3-10 VRIAGDYYYYYGMDV 37D3 155 V_(H)15 CDRH3-11 DRVLSYYAMAV39F11 156 V_(H)17 CDRH3-12 DRAAAGLHYYYGMDV 39F7 V_(H)16 39G5 V_(H)1840D2 157 V_(H)14 CDRH3-13 ENIVVIPAAIFAGWFDP

TABLE 3B Exemplary CDRL Sequences SEQ Contained ID in Clone NO:Reference Designation Sequence 20D4 158 V_(L)12 CDRL1-1 RASQDIRYDLG18B11.1 159 V_(L)10 CDRL1-2 RSSQSLLYYNGFTYLD 12C11 160 V_(L)8 CDRL1-3RASQNFDSSSLA 18G1 161 V_(L)4 CDRL1-4 RASQNFDSSYLA 17D8 162 V_(L)5CDRL1-5 RASQSVSGNYLA 26H11 V_(L)6 21B4 163 V_(L)9 CDRL1-6 RASQSVSSTYLA21H2 V_(L)9 39F7 V_(L)16 39F11 V_(L)17 39G5 V_(L)18 12E4 164 V_(L)7CDRL1-7 RASQNFDSNYLA 18B11.2 165 V_(L)11 CDRL1-8 RASQSVNSNLA 16H7 166V_(L)3 CDRL1-9 GGNNIGSESVH 24H11 V_(L)3 22H5 167 V_(L)2 CDRL1-10GGNNIGSQSVH 17C3 V_(L)1 46D11 168 V_(L)13 CDRL1-11 RASQGISIWLA 40D2 169V_(L)14 CDRL1-12 KSSQSLLQSDGKTYLY 37D3 170 V_(L)15 CDRL1-13RSSQSLLHSNGYNFLD 20D4 171 V_(L)12 CDRL2-1 AASSLQS 46D11 V_(L)13 18B11.1172 V_(L)10 CDRL2-2 LGSNRAS 12C11 173 V_(L)8 CDRL2-3 GASSRAT 17D8 V_(L)521B4 V_(L)9 21H2 V_(L)9 26H11 V_(L)6 12E4 V_(L)7 39F7 V_(L)16 39F11V_(L)17 18G1 174 V_(L)4 CDRL2-4 GTSSRAT 18B11.2 175 V_(L)11 CDRL2-5GVSTRAT 16H7 176 V_(L)3 CDRL2-6 DDSDRPS 24H11 V_(L)3 22H5 V_(L)2 17C3V_(L)1 40D2 177 V_(L)14 CDRL2-7 EVSNRFS 37D3 178 V_(L)15 CDRL2-8 LGSDRAS39G5 179 V_(L)18 CDRL2-9 GASFRAT 20D4 180 V_(L)12 CDRL3-1 LQHNSYPLT18B11.1 181 V_(L)10 CDRL3-2 MQSLQTPFT 12C11 182 V_(L)8 CDRL3-3 QQCGSSPLT18G1 183 V_(L)4 CDRL3-4 QQYGGSPLT 17D8 184 V_(L)5 CDRL3-5 QQYGSAPLT 21B4185 V_(L)9 CDRL3-6 QQYGSSFT 21H2 V_(L)9 26H11 186 V_(L)6 CDRL3-7QQYGSSPLT 12E4 V_(L)7 18B11.2 187 V_(L)11 CDRL3-8 QQYNNWPPT 16H7 188V_(L)3 CDRL3-9 QVWDGNSDHVV 24H11 V_(L)3 22H5 189 V_(L)2 CDRL3-10QVWDNTSDHVV 17C3 190 V_(L)1 CDRL3-11 QVWDSSSDHVV 46D11 191 V_(L)13CDRL3-12 QQANDFPIT 40D2 192 V_(L)14 CDRL3-13 MQSIQLPRT 37D3 193 V_(L)15CDRL3-14 MQALQTPCS 39F7 194 V_(L)16 CDRL3-15 QQSGSSPLT 39F11 V_(L)1739G5 V_(L)18

TABLE 3C Coding Sequences for CDRHs SEQ Contained ID in Clone NO:Reference Designation Sequence 20D4 195 V_(H)12 CDRH GATTTATCCATGCAC 1-117C3 196 V_(H)1 CDRH AATGCTAGAATGGGTGTGAGC 22H5 V_(H)2 1-2 16H7 V_(H)324H11 V_(H)4 18B11.1 197 V_(H)11 CDRH GACGCCTGGATGAGC 18B11.2 V_(H)111-3 18G1 198 V_(H)5 CDRH ACCTATGCCATGAGC 12C11 V_(H)8 1-4 12E4 V_(H)817D8 V_(H)6 26H11 V_(H)7 21B4 199 V_(H)10 CDRH AGTTACTTCTGGAGC 21H2V_(H)9 1-5 46D11 200 V_(H)13 CDRH AATGCTAGAATGGGTGTGAAC 1-6 37D3 201V_(H)15 CDRH AACGCCTGGATGAGC 1-7 39F11 202 V_(H)17 CDRH AGCTATGGCATCCAC39G5 V_(H)18 1-8 39F7 203 V_(H)16 CDRH AACTATGGCATTCAC 1-9 40D2 204V_(H)14 CDRH AGTGGTGGTTACAACTGGAGC 1-10 20D4 205 V_(H)12 CDRHGGTTTTGATCCTGAAGATGGTGAAACAATCT 2-1 ACGCACAGAAGTTCCAGGGC 17C3 206 V_(H)1CDRH CACATTTTTTCGAATGACGAAAAA 22H5 V_(H)2 2-2 TCCTACAGCACATCTCTGAAGAGC16H7 V_(H)3 46D11 V_(H)13 24H11 207 V_(H)4 CDRHCACATTTTTTCGAATGACGAAAAATC 2-3 CTACAGCACATCTCTGAAGAAC 18B11.1 208V_(H)11 CDRH CGTATTAAAAGCAAAACTGATGGTGGGA 18B11.2 V_(H)11 2-4CAACAGACTACGCTGCACCCGTGAAAGGC 37D3 V_(H)15 18G1 209 V_(H)5 CDRHGGTATTAGTGGTAGTGGTGTCAGCACACA 2-5 CTACGCAGACTCCGTGAAGGGC 12C11 210V_(H)8 CDRH GGTATTAGTGGTAGTGGTGTTAGCACATAC 12E4 V_(H)8 2-6TACGCAGACTCCGTGAAGGGC 17D8 211 V_(H)6 CDRHGCTATCAGTGGTAGTGGTGTTAGCACATAC 2-7 TACGCAGACTCCGTGAAGGGC 26H11 212V_(H)7 CDRH GCTATTAGTGGCAGTGGTGTGAGCACAAAC 2-8 TACGCAGACTCCGTGAAGGGC21B4 213 V_(H)10 CDRH CGTATCTATACCAGTGGGAGCACCAACTACA 21H2 V_(H)9 2-9ACCCCTCCCTCAAGAGT 39F11 214 V_(H)17 CDRHGTTATATGGTATGATGGAAGTGATAAATACTA 2-10 TGCAGACTCCGTGAAGGGC 39F7 215V_(H)16 CDRH GTTATATGGTATGATGGAAGTATTAAATACTA 2-11 TGCAGACTCCGTGAAGGGC39G5 216 V_(H)18 CDRH GTTATATGGTATGATGGAAGTGATAAATACTA 2-12TGGAGACTCCGTGAAGGGC 40D2 217 V_(H)14 CDRHAACATCTATTACAGTGGGAGCACCTACTACAA 2-13 CCCGTCCCTCAAGAGT 20D4 218 V_(H)12CDRH ATTGTAGTAGTCCCAGCTGCTATACAGAGTTA 3-1 CTACTACTACTACGGTATGGGCGTC 17C3219 V_(H)1 CDRH ATATTATTACTGGGAGCTTACTACTACTACGG 3-2 TATGGACGTC 22H5 220V_(H)2 CDRH ATATTATTAGTGGGAGCTTACTACTACTGCGG 3-3 TATGGACGTC 16H7 221V_(H)3 CDRH TCAGTAGTAACTGGCGGCTACTACTACGACG 24H11 V_(H)4 3-4 GTATGGACGTC18B11.1 222 V_(H)11 CDRH ACGTATAGCAGTGGCTGGTACGTATGGGAC 18B11.2 V_(H)113-5 TACTACGGTATGGACGTC 18G1 223 V_(H)5 CDRHTCCCTCATTGTAGTAATAGTATATGCCCTTG 3-6 ACCAC 12C11 224 V_(H)8 CDRHTCCCTTATTGTAGTAATAGTATATGCCCT 12E4 V_(H)8 3-7 TGACTAC 17D8 225 V_(H)6CDRH TCCCTTATTGTAGTAATGGTGTATGTCCT 26H11 V_(H)7 3-8 TGACTAC 21B4 226V_(H)10 CDRH GATCCGGACGGTGACTACTACTACTACG 21H2 V_(H)9 3-9 GTATGGACGTC46D11 227 V_(H)13 CDRH GTTCGTATAGCAGGTGATTACTACTACTA 3-10CTACGGTATGGACGTC 37D3 228 V_(H)15 CDRH GATCGGGTGCTAAGCTACTACGCTATGG 3-11CCGTC 39F11 229 V_(H)17 CDRH GATAGGGCAGCAGCTGGTCTCCACTATT 39F7 V_(H)163-12 ATTACGGTATGGACGTC 39G5 V_(H)18 40D2 230 V_(H)14 CDRHGAGAATATTGTAGTAATACCAGCTGCTAT 3-13 ATTCGCGGGTTGGTTCGACCCC

TABLE 3D Coding Sequences for CDRLs SEQ ID Contained in Clone NO:Reference Designation Sequence 20D4 231 V_(L)12 CDRL1-1CGGGCAAGTCAGGACATTAGATATGATT TAGGC 18B11.1 232 V_(L)10 CDRL1-2AGGTCTAGTCAGAGCCTCCTGTATTATA ATGGATTCACCTATTTGGAT 12C11 233 V_(L)8CDRL1-3 AGGGCCAGTCAGAATTTTGACAGCAGC TCCTTAGCC 18G1 234 V_(L)4 CDRL1-4AGGGCCAGTCAGAATTTTGACAGCAGT TACTTAGCC 17D8 235 V_(L)5 CDRL1-5AGGGCCAGTCAGAGTGTTAGCGGCAAC 26H11 V_(L)6 TACTTGGCC 21B4 236 V_(L)9CDRL1-6 AGGGCCAGTCAGAGTGTGAGCAGTACC 21H2 V_(L)9 TACTTAGCC 39F7 V_(L)1639F11 V_(L)17 39G5 V_(L)18 12E4 237 V_(L)7 CDRL1-7AGGGCCAGTCAGAATTTCGACAGCAAC TACTTAGCC 18B11.2 238 V_(L)11 CDRL1-8AGGGCCAGTCAGAGTGTTAACAGCAAC TTAGCC 16H7 239 V_(L)3 CDRL1-9GGGGGAAACAACATTGGAAGTGAAAGTG 24H11 V_(L)3 TGCAC 22H5 240 V_(L)2 CDRL1-10GGGGGAAACAACATTGGAAGTCAAAGTG 17C3 V_(L)1 TGCAC 46D11 241 V_(L)13CDRL1-11 CGGGCGAGTCAGGGTATTAGCATCTGGT TAGCC 40D2 242 V_(L)14 CDRL1-12AAGTCTAGTCAGAGCCTCCTACAGAGTG ATGGAAAGACCTATTTGTAT 37D3 243 V_(L)15CDRL1-13 AGGTCTAGTCAGAGCCTCCTGCATAGTA ATGGATACAACTTTTTGGAT 20D4 244V_(L)12 CDRL2-1 GCTGCATCCAGTTTGCAAAGT 46D11 V_(L)13 18B11.1 245 V_(L)10CDRL2-2 TTGGGTTCTAATCGGGCCTCC 12C11 246 V_(L)8 CDRL2-3GGTGCATCCAGCAGGGCCACT 17D8 V_(L)5 21B4 V_(L)9 21H2 V_(L)9 26H11 V_(L)612E4 V_(L)7 39F7 V_(L)16 39F11 V_(L)17 18G1 247 V_(L)4 CDRL2-4GGTACATCCAGCAGGGCCACT 18B11.2 248 V_(L)11 CDRL2-5 GGTGTATCCACCAGGGCCACT16H7 249 V_(L)3 CDRL2-6 GATGATAGCGACCGGCCCTCA 24H11 V_(L)3 22H5 V_(L)217C3 V_(L)1 40D2 250 V_(L)14 CDRL2-7 GAAGTTTCCAACCGATTCTCT 37D3 251V_(L)15 CDRL2-8 TTGGGTTCTGATCGGGCCTCC 20D4 252 V_(L)12 CDRL3-1CTACAGCATAATAGTTACCCTCTCACT 18B11.1 253 V_(L)10 CDRL3-2ATGCAGTCTCTGCAAACTCCATTCACT 12C11 254 V_(L)8 CDRL3-3CAGCAGTGTGGTAGCTCACCGCTCACT 18G1 255 V_(L)4 CDRL3-4CAGCAGTATGGTGGCTCACCGCTCACT 17D8 256 V_(L)5 CDRL3-5CAGCAGTATGGTAGCGCACCGCTCACT 21B4 257 V_(L)9 CDRL3-6CAGCAGTATGGAAGTTCATTCACT 21H2 V_(L)9 26H11 258 V_(L)6 CDRL3-7CAGCAGTATGGTAGCTCACCGCTCACT 12E4 V_(L)7 18B11.2 259 V_(L)11 CDRL3-8CAGCAGTATAATAACTGGCCTCCGACG 16H7 260 V_(L)3 CDRL3-9CAGGTGTGGGATGGTAATAGTGATCAT 24H11 V_(L)3 GTGGTA 22H5 261 V_(L)2 CDRL3-10CAGGTGTGGGATAATACTAGTGATCAT GTGGTA 17C3 262 V_(L)1 CDRL3-11CAGGTGTGGGATAGTAGTAGTGATCAT GTGGTA 46D11 263 V_(L)13 CDRL3-12CAACAGGCTAACGATTTCCCGATCACC 40D2 264 V_(L)14 CDRL3-13ATGCAAAGTATACAGCTTCCTCGGACG 37D3 265 V_(L)15 CDRL3-14ATGCAAGCTCTACAAACTCCGTGCAGT 39F7 266 V_(L)16 CDRL3-15CAGCAGTCTGGTAGCTCACCTCTCACT 39F11 V_(L)17 39G5 V_(L)18

The structure and properties of CDRs within a naturally occurringantibody has been described, supra. Briefly, in a traditional antibody,the CDRs are embedded within a framework in the heavy and light chainvariable region where they constitute the regions responsible forantigen binding and recognition. A variable region comprises at leastthree heavy or light chain CDRs, see, supra (Kabat et al., 1991,Sequences of Proteins of Immunological Interest, Public Health ServiceN.I.H., Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol.196:901-917; Chothia et al., 1989, Nature 342: 877-883), within aframework region (designated framework regions 1-4, FR1, FR2, FR3, andFR4, by Kabat et al., 1991; see also Chothia and Lesk, 1987, supra). TheCDRs provided herein, however, can not only be used to define theantigen binding domain of a traditional antibody structure, but can beembedded in a variety of other polypeptide structures, as describedherein.

In one aspect, the CDRs provided are (a) a CDRH selected from the groupconsisting of (i) a CDRH1 selected from the group consisting of SEQ IDNO:121-131; (ii) a CDRH2 selected from the group consisting of SEQ IDNO:132-144; (iii) a CDRH3 selected from the group consisting of SEQ IDNO:145-157; and (iv) a CDRH of (i), (ii) and (iii) that contains one ormore amino acid substitutions, deletions or insertions of no more thanfive, four, three, two, or one amino acids; (B) a CDRL selected from thegroup consisting of (i) a CDRL1 selected from the group consisting ofSEQ ID NO:158-170; (ii) a CDRL2 selected from the group consisting ofSEQ ID NO:171-179; (iii) a CDRL3 selected from the group consisting ofSEQ ID NO:180-194; and (iv) a CDRL of (i), (ii) and (iii) that containsone or more amino acid substitutions, deletions or insertions of no morethan 1, 2, 3, 4, or 5 amino acids amino acids.

In another aspect, an antigen binding protein comprises 1, 2, 3, 4, 5,or 6 variant forms of the CDRs listed in Tables 3A and 3B and Table 6C,infra, each having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to a CDR sequence listed in Tables 3A and 3B and Table6C, infra. Some antigen binding proteins comprise 1, 2, 3, 4, 5, or 6 ofthe CDRs listed in Tables 3A and 3B and Table 6C, infra, each differingby no more than 1, 2, 3, 4 or 5 amino acids from the CDRs listed inthese tables.

In still another aspect, an antigen binding protein includes thefollowing associations of CDRL1, CDRL2 and CDRL3: SEQ ID NOs:167, 176,and 190; SEQ ID NOs:167, 176, and 189, SEQ ID NOs:166, 176, and 188; SEQID NOs:166, 176, and 188; SEQ ID NOs:161, 174, and 183; SEQ ID NOs:162,173, and 184; SEQ ID NOs:162, 173, and 186; SEQ ID NOs:164, 173, and186; SEQ ID NOs:160, 173, and 182; SEQ ID NOs:163, 173, and 185; SEQ IDNOs:163, 173, and 185; SEQ ID NOs:159, 172, and 181; SEQ ID NOs:165,175, and 187; SEQ ID NOs:158, 171, and 180; SEQ ID NOs:168, 171, and191; SEQ ID NOs:169, 177 and 192; SEQ ID NOs:170, 178, and 193; SEQ IDNOs:163, 173, and 194; SEQ ID NOs:163, 173 and 194; and SEQ ID NOs:163,179, and 194.

In an additional aspect, an antigen binding protein includes thefollowing associations of CDRH1, CDRH2 and CDRH3: SEQ ID NOs:122, 133,and 146; SEQ ID NOs:122, 133, and 147; SEQ ID NOs:122, 133, and 148; SEQID NOs:122, 134, and 148; SEQ ID NOs:124, 136, and 150; SEQ ID NOs:124,138, and 152; SEQ ID NOs:124, 139, and 152; SEQ ID NOs:124, 137, and151; SEQ ID NOs:124, 137, and 151; SEQ ID NOs:131, 140, and 153; SEQ IDNOs:125, 140, and 153; SEQ ID NOs:123, 135, and 149; SEQ ID NOs:123,135, and 149; SEQ ID NOs:121, 132, and 145; SEQ ID NOs:126, 133, and154; SEQ ID NOs:130, 144, and 157; SEQ ID NOs:127, 135, and 155; SEQ IDNOs:129, 142, and 156; SEQ ID NOs:128, 141, and 156; and SEQ ID NOs:128,143, and 156.

In another aspect, an antigen binding protein includes the followingassociations of CDRL1, CDRL2 and CDRL3 with CDRH1, CDRH2 and CDRH3: SEQID NOs:167, 176, and 190; SEQ ID NOs:167, 176, and 189, SEQ ID NOs:166,176, and 188; SEQ ID NOs:166, 176, and 188; SEQ ID NOs:161, 174, and183; SEQ ID NOs:162, 173, and 184; SEQ ID NOs:162, 173, and 186; SEQ IDNOs:164, 173, and 186; SEQ ID NOs:160, 173, and 182; SEQ ID NOs:163,173, and 185; SEQ ID NOs:163, 173, and 185; SEQ ID NOs:159, 172, and181; SEQ ID NOs:165, 175, and 187; SEQ ID NOs:158, 171, and 180; SEQ IDNOs:168, 171, and 191; SEQ ID NOs:169, 177 and 192; SEQ ID NOs:170, 178,and 193; SEQ ID NOs:163, 173, and 194; SEQ ID NOs:163, 173 and 194; SEQID NOs:163, 179, and 194 with SEQ ID NOs:122, 133, and 146; SEQ IDNOs:122, 133, and 147; SEQ ID NOs:122, 133, and 148; SEQ ID NOs:122,134, and 148; SEQ ID NOs:124, 136, and 150; SEQ ID NOs:124, 138, and152; SEQ ID NOs:124, 139, and 152; SEQ ID NOs:124, 137, and 151; SEQ IDNOs:124, 137, and 151; SEQ ID NOs:131, 140, and 153; SEQ ID NOs:125,140, and 153; SEQ ID NOs:123, 135, and 149; SEQ ID NOs:123, 135, and149; SEQ ID NOs:121, 132, and 145; SEQ ID NOs:126, 133, and 154; SEQ IDNOs:130, 144, and 157; SEQ ID NOs:127, 135, and 155; SEQ ID NOs:129,142, and 156; SEQ ID NOs:128, 141, and 156; and SEQ ID NOs:128, 143, and156.

Consensus Sequences

In yet another aspect, the CDRs disclosed herein include consensussequences derived from groups of related monoclonal antibodies. Asdescribed herein, a “consensus sequence” refers to amino acid sequenceshaving conserved amino acids common among a number of sequences andvariable amino acids that vary within a given amino acid sequences. TheCDR consensus sequences provided include CDRs corresponding to each ofCDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3.

Consensus sequences were determined using standard analyses of the CDRscorresponding to the V_(H) and V_(L) of the disclosed antibodies, someof which specifically bind (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c orFGFR4; or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c,FGFR3c, and FGFR4. The consensus sequences were determined by keepingthe CDRs contiguous within the same sequence corresponding to a V_(H) orV_(L).

Light Chain CDR3

Group 1 LQHNSYPLT (SEQ ID NO: 267) Group 2 MQSLQTPFT (SEQ ID NO: 268)Group 3 QQYNNWPPT (SEQ ID NO: 269) Group 4 MQSIQLPRT (SEQ ID NO: 270)Group 5 QQANDFPIT (SEQ ID NO: 271) Group 6 MQALQTPCS (SEQ ID NO: 272)Group 7 QVWD G N SDHVV (SEQ ID NO: 273) QVWD N T SDHVV (SEQ ID NO: 274)QVWD S S SDHVV (SEQ ID NO: 275) QVWD X ₁ X ₂ SDHVV (SEQ ID NO: 276)wherein X₁ is G, S or N and X₂ is S, T or N. Group 8 QQ C G S S P L T(SEQ ID NO: 277) QQ Y G G S P L T (SEQ ID NO: 278) QQ Y G S A P L T (SEQID NO: 279) QQ Y G S S F T (SEQ ID NO: 280) QQ Y G S S P L T (SEQ ID NO:281) QQ S G S S P L T (SEQ ID NO: 282) QQ X ₃ G X ₄ X ₅ X ₆ X ₇ T (SEQID NO: 283) wherein X₃ is C, Y or S, X₄ is S or G, X₅ is S or A, X₆ is Por F and X₇ is L or absent.Light Chain CDR2

Group 1 AASSLQS (SEQ ID NO: 284) Group 2 GVSTRAT (SEQ ID NO: 285) Group3 DDSDRPS (SEQ ID NO: 286) Group 4 EVSNRFS (SEQ ID NO: 287) Group 5 L GS N R A S (SEQ ID NO: 288) L G S D R A S (SEQ ID NO: 289) L G S X ₂₇ R AS (SEQ ID NO: 290) wherein X₂₇ is N or D. Group 6 G A S S RAT (SEQ IDNO: 291) G T S S RAT (SEQ ID NO: 292) G A S F RAT (SEQ ID NO: 293) G X ₈S X ₂₈ RAT (SEQ ID NO: 294) wherein X₈ is A or T and X₂₈ is S or F.Light Chain CDR1

Group 1 RASQSVNSNLA (SEQ ID NO: 295) Group 2 RASQDIRYDLG (SEQ ID NO:296) Group 3 RASQGISIWLA (SEQ ID NO: 297) Group 4 KSSQSLLQSDGKTYLY (SEQID NO: 298) Group 5 RASQ N F D S S S LA (SEQ ID NO: 299) RASQ N F D S SY LA (SEQ ID NO: 300) RASQ S V S G N Y LA (SEQ ID NO: 301) RASQ S V S GT Y LA (SEQ ID NO: 302) RASQ N F D S N Y LA (SEQ ID NO: 303) RASQ X ₉ X₁₀ X ₁₁ X ₁₂ X ₁₃ X ₁₄ LA (SEQ ID NO: 304) wherein X₉ is A or S, X₁₀ isV or F, X₁₁ is D or S, X₁₂ is G or S, X₁₃ is S, N or T, and X₁₄ is S orY. Group 6 GGNNIGS E SVH (SEQ ID NO: 305) GGNNIGS Q SVH (SEQ ID NO: 306)GGNNIGS X ₁₅ SVH (SEQ ID NO: 307) wherein X₁₅ is E or Q. Group 7 RSSQSLLY Y NG F T Y LD (SEQ ID NO: 308) RSSQSLL H S NG Y N F LD (SEQ ID NO:309) RSSQSLL X ₂₉ X ₃₀ NG X ₃₁ X ₃₂ X ₃₃ LD (SEQ ID NO: 310) wherein X₂₉is Y or H, X₃₀ is Y or S, X₃₁ is F or Y, X₃₂ is T or N and X₃₃ is Y orF.Heavy CDR3

Group 1 IVVVPAAIQSYYYYYGMGV (SEQ ID NO: 311) Group 2 DPDGDYYYYGMDV (SEQID NO: 312) Group 3 TYSSGWYVWDYYGMDV (SEQ ID NO: 313) Group 4DRVLSYYAMAV (SEQ ID NO: 314) Group 5 VRIAGDYYYYYGMDV (SEQ ID NO: 315)Group 6 ENIVVIPAAIFAGWFDP (SEQ ID NO: 316) Group 7 DRAAAGLHYYYGMDV (SEQID NO: 317) Group 8 I L L L G A YYY Y GMDV (SEQ ID NO: 318) I L L V G AYYY C GMDV (SEQ ID NO: 319) V V T G G YYY D GMDV (SEQ ID NO: 320) S V VT G G YYY D GMDV (SEQ ID NO: 321) X ₃₄ X ₁₆ X ₁₇ X ₁₈ G X ₁₉ YYY X ₂₀GMDV (SEQ ID NO: 322) Wherein X₃₄ is I, V or S, X₁₆ is L or V, X₁₇ is L,T or V, X₁₈ is L, V, G or T, X₁₉ is A, G or absent and X₂₀ is Y, C or D.Group 9 SLIVV I VY A LD H (SEQ ID NO: 323) SLIVV I VY A LD Y (SEQ ID NO:324) SLIVV M VY V LD Y (SEQ ID NO: 325) SLIVV X ₂₁ VY X ₂₂ LD X ₂₃ (SEQID NO: 326) Wherein X₂₁ is I or M, X₂₂ is A or V and X₂₃ is H or Y.Heavy CDR2

Group 1 GFDPEDGETIYAQKFQG (SEQ ID NO: 327) Group 2 RIKSK T DGGTTDYAAPVKG(SEQ ID NO: 328) RIKSK DGGTTDYAAPVKG (SEQ ID NO: 330) RIKSK X ₄₂DGGTTDYAAPVKG (SEQ ID NO: 483) wherein X₄₂ is T or absent. Group 3HIFSNDEKSYSTSLK S (SEQ ID NO: 331) HIFSNDEKSYSTSLK N (SEQ ID NO: 332)HIFSNDEKSYSTSLK X ₂₄ (SEQ ID NO: 333) wherein X₂₄ is S or N. Group 4 GISGSGVST H YADSVKG (SEQ ID NO: 334) G ISGSGVST Y YADSVKG (SEQ ID NO:335) A ISGSGVST Y YADSVKG (SEQ ID NO: 336) A ISGSGVST N YADSVKG (SEQ IDNO: 337) X ₂₅ ISGSGVST X ₂₆ YADSVKG (SEQ ID NO: 338) wherein X₂₅ is G orA and X₂₆ is H, Y or N. Group 5 VIWYDGS D KYY A DSVKG (SEQ ID NO: 339)VIWYDGS I KYY G DSVKG (SEQ ID NO: 340) VIWYDGS X ₃₅ KYY X ₃₆ DSVKG (SEQID NO: 341) wherein X₃₅ is D or I and X₃₆ is A or G. Group 6 N IY Y SGSTY YNPSLKS (SEQ ID NO: 342) R IY T SGST Y YNPSLKS (SEQ ID NO: 343) R IY TSGST N YNPSLKS (SEQ ID NO: 329) X ₃₇ IY X ₃₈ SGST X ₄₁ YNPSLKS (SEQ IDNO: 344) wherein X₃₇ is N or R, X₃₈ is Y or T and X₄₁ is Y or N.Heavy CDR1

Group 1 DLSMH (SEQ ID NO: 345) Group 2 DAWMS (SEQ ID NO: 346) Group 3TYAMS (SEQ ID NO: 347) Group 4 SYFWS (SEQ ID NO: 348) Group 5 SGGYNWS(SEQ ID NO: 349) Group 6 NARMGV S (SEQ ID NO: 350) NARMGV N (SEQ ID NO:351) NARMGV X ₃₉ (SEQ ID NO: 352) wherein X₃₉ is S or N. Group 7 S YGIH(SEQ ID NO: 353) N YGIH (SEQ ID NO: 354) X ₄₀ YGIH (SEQ ID NO: 355)wherein X₄₀ is S or N.

In some cases an antigen binding protein comprises at least one heavychain CDR1, CDR2, or CDR3 having one of the above consensus sequences.In some cases, an antigen binding protein comprises at least one lightchain CDR1, CDR2, or CDR3 having one of the above consensus sequences.In other cases, the antigen binding protein comprises at least two heavychain CDRs according to the above consensus sequences, and/or at leasttwo light chain CDRs according to the above consensus sequences. Instill other cases, the antigen binding protein comprises at least threeheavy chain CDRs according to the above consensus sequences, and/or atleast three light chain CDRs according to the above consensus sequences.

Exemplary Antigen Binding Proteins

According to one aspect, an isolated antigen binding protein comprising(a) one or more heavy chain complementary determining regions (CDRHs)selected from the group consisting of: (i) a CDRH1 selected from thegroup consisting of SEQ ID NO:121-131; (ii) a CDRH2 selected from thegroup consisting of SEQ ID NO:132-144; (iii) a CDRH3 selected from thegroup consisting of SEQ ID NO:145-157; and (iv) a CDRH of (i), (ii) and(iii) that contains one or more amino acid substitutions, deletions orinsertions of no more than 1, 2, 3, 4, or 5 amino acids; (b) one or morelight chain complementary determining regions (CDRLs) selected from thegroup consisting of: (i) a CDRL1 selected from the group consisting ofSEQ ID NO:158-170; (ii) a CDRL2 selected from the group consisting ofSEQ ID NO:171-179; (iii) a CDRL3 selected from the group consisting ofSEQ ID NO:180-194; and (iv) a CDRL of (i), (ii) and (iii) that containsone or more amino acid substitutions, deletions or insertions of no morethan five, four, three, four, two or one amino acids; or (c) one or moreheavy chain CDRHs of (a) and one or more light chain CDRLs of (b).

In another embodiment, the CDRHs have at least 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% sequence identity with an amino acid sequenceselected from the group consisting of SEQ ID NO:121-157, and/or theCDRLs have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%sequence identity with an amino acid sequence selected from the groupconsisting of SEQ ID NO:158-194. In a further embodiment, the VH isselected from the group consisting of SEQ ID NO:121-157, and/or the VLis selected from the group consisting of SEQ ID NO: 158-194.

According to one aspect, an isolated antigen binding protein comprising(a) one or more variable heavy chains (VHs) selected from the groupconsisting of: (i) SEQ ID NO:121-157; and (ii) a VH of (i) that containsone or more amino acid substitutions, deletions or insertions of no morethan five, four, three, four, two or one amino acids; (b) one or morevariable light chains (VLs) selected from the group consisting of: (i)SEQ ID NO:158-194, and (ii) a VL of (i) that contains one or more aminoacid substitutions, deletions or insertions of no more than five, four,three, four, two or one amino acids; or (c) one or more variable heavychains of (a) and one or more variable light chains of (b).

In another embodiment, the variable heavy chain (VH) has at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with anamino acid sequence selected from the group consisting of SEQ IDNO:121-157, and/or the variable light chain (VL) has at least 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with an aminoacid sequence selected from the group consisting of SEQ ID NO: 158-194.

In one aspect, also provided is an antigen binding protein thatspecifically binds to an epitope comprising one or more amino acidresidues from FGFR1c, FGRF2c, FGFR3c, and FGFR4.

In one aspect, also provided is an antigen binding protein thatspecifically binds to an epitope comprising one or more amino acidresidues from β-Klotho.

In another aspect, also provided is an isolated antigen binding proteinthat specifically binds to an epitope comprising one or more amino acidresidues from both β-Klotho and one or more amino acid residues fromFGFR1c, FGFR2c, FGFR3c, or FGFR4.

In yet another embodiment, the isolated antigen binding proteindescribed hereinabove comprises a first amino acid sequence comprisingat least one of the CDRH consensus sequences disclosed herein, and asecond amino acid sequence comprising at least one of the CDRL consensussequences disclosed herein.

In one aspect, the first amino acid sequence comprises at least two ofthe CDRH consensus sequences, and/or the second amino acid sequencecomprises at least two of the CDRL consensus sequences. In certainembodiments, the first and the second amino acid sequence are covalentlybonded to each other.

In a further embodiment, the first amino acid sequence of the isolatedantigen binding protein comprises the CDRH3 of SEQ ID NO:146, the CDRH2of SEQ ID NO:133, and the CDRH1 of SEQ ID NO:122, and/or the secondamino acid sequence of the isolated antigen binding protein comprisesthe CDRL3 of SEQ ID NO:190, the CDRL2 of SEQ ID NO:176, and the CDRL1 ofSEQ ID NO:167.

In a further embodiment, the first amino acid sequence of the isolatedantigen binding protein comprises the CDRH3 of SEQ ID NO:147, the CDRH2of SEQ ID NO:133, and the CDRH1 of SEQ ID NO:122, and/or the secondamino acid sequence of the isolated antigen binding protein comprisesthe CDRL3 of SEQ ID NO:189, the CDRL2 of SEQ ID NO:176, and the CDRL1 ofSEQ ID NO:167.

In a further embodiment, the first amino acid sequence of the isolatedantigen binding protein comprises the CDRH3 of SEQ ID NO:148, the CDRH2of SEQ ID NO:133, and the CDRH1 of SEQ ID NO:122, and/or the secondamino acid sequence of the isolated antigen binding protein comprisesthe CDRL3 of SEQ ID NO:188, the CDRL2 of SEQ ID NO:176, and the CDRL1 ofSEQ ID NO:166.

In a further embodiment, the first amino acid sequence of the isolatedantigen binding protein comprises the CDRH3 of SEQ ID NO:148, the CDRH2of SEQ ID NO:134, and the CDRH1 of SEQ ID NO:122, and/or the secondamino acid sequence of the isolated antigen binding protein comprisesthe CDRL3 of SEQ ID NO:188, the CDRL2 of SEQ ID NO:176, and the CDRL1 ofSEQ ID NO:166.

In a further embodiment, the first amino acid sequence of the isolatedantigen binding protein comprises the CDRH3 of SEQ ID NO:150, the CDRH2of SEQ ID NO:136, and the CDRH1 of SEQ ID NO:124, and/or the secondamino acid sequence of the isolated antigen binding protein comprisesthe CDRL3 of SEQ ID NO:183, the CDRL2 of SEQ ID NO:174, and the CDRL1 ofSEQ ID NO:161.

In a further embodiment, the first amino acid sequence of the isolatedantigen binding protein comprises the CDRH3 of SEQ ID NO:152, the CDRH2of SEQ ID NO:138, and the CDRH1 of SEQ ID NO:124, and/or the secondamino acid sequence of the isolated antigen binding protein comprisesthe CDRL3 of SEQ ID NO:184, the CDRL2 of SEQ ID NO:173, and the CDRL1 ofSEQ ID NO:162.

In a further embodiment, the first amino acid sequence of the isolatedantigen binding protein comprises the CDRH3 of SEQ ID NO:152, the CDRH2of SEQ ID NO:139, and the CDRH1 of SEQ ID NO:124, and/or the secondamino acid sequence of the isolated antigen binding protein comprisesthe CDRL3 of SEQ ID NO:186, the CDRL2 of SEQ ID NO:173, and the CDRL1 ofSEQ ID NO:162.

In a further embodiment, the first amino acid sequence of the isolatedantigen binding protein comprises the CDRH3 of SEQ ID NO:151, the CDRH2of SEQ ID NO:137, and the CDRH1 of SEQ ID NO:124, and/or the secondamino acid sequence of the isolated antigen binding protein comprisesthe CDRL3 of SEQ ID NO:186, the CDRL2 of SEQ ID NO:173, and the CDRL1 ofSEQ ID NO:164.

In a further embodiment, the first amino acid sequence of the isolatedantigen binding protein comprises the CDRH3 of SEQ ID NO:151, the CDRH2of SEQ ID NO:137, and the CDRH1 of SEQ ID NO:124, and/or the secondamino acid sequence of the isolated antigen binding protein comprisesthe CDRL3 of SEQ ID NO:182, the CDRL2 of SEQ ID NO:173, and the CDRL1 ofSEQ ID NO:160.

In a further embodiment, the first amino acid sequence of the isolatedantigen binding protein comprises the CDRH3 of SEQ ID NO:153, the CDRH2of SEQ ID NO:140, and the CDRH1 of SEQ ID NO:131, and/or the secondamino acid sequence of the isolated antigen binding protein comprisesthe CDRL3 of SEQ ID NO:185, the CDRL2 of SEQ ID NO:173, and the CDRL1 ofSEQ ID NO:163.

In a further embodiment, the first amino acid sequence of the isolatedantigen binding protein comprises the CDRH3 of SEQ ID NO:153, the CDRH2of SEQ ID NO:140, and the CDRH1 of SEQ ID NO:125, and/or the secondamino acid sequence of the isolated antigen binding protein comprisesthe CDRL3 of SEQ ID NO:185, the CDRL2 of SEQ ID NO:173, and the CDRL1 ofSEQ ID NO:163.

In a further embodiment, the first amino acid sequence of the isolatedantigen binding protein comprises the CDRH3 of SEQ ID NO:149, the CDRH2of SEQ ID NO:135, and the CDRH1 of SEQ ID NO:123, and/or the secondamino acid sequence of the isolated antigen binding protein comprisesthe CDRL3 of SEQ ID NO:181, the CDRL2 of SEQ ID NO:172, and the CDRL1 ofSEQ ID NO:159.

In a further embodiment, the first amino acid sequence of the isolatedantigen binding protein comprises the CDRH3 of SEQ ID NO:149, the CDRH2of SEQ ID NO:135, and the CDRH1 of SEQ ID NO:123, and/or the secondamino acid sequence of the isolated antigen binding protein comprisesthe CDRL3 of SEQ ID NO:187, the CDRL2 of SEQ ID NO:175, and the CDRL1 ofSEQ ID NO:165.

In a further embodiment, the first amino acid sequence of the isolatedantigen binding protein comprises the CDRH3 of SEQ ID NO:145, the CDRH2of SEQ ID NO:132, and the CDRH1 of SEQ ID NO:121, and/or the secondamino acid sequence of the isolated antigen binding protein comprisesthe CDRL3 of SEQ ID NO:180, the CDRL2 of SEQ ID NO:171, and the CDRL1 ofSEQ ID NO:158.

In a further embodiment, the first amino acid sequence of the isolatedantigen binding protein comprises the CDRH3 of SEQ ID NO:154, the CDRH2of SEQ ID NO:133, and the CDRH1 of SEQ ID NO:126, and/or the secondamino acid sequence of the isolated antigen binding protein comprisesthe CDRL3 of SEQ ID NO:191, the CDRL2 of SEQ ID NO:171, and the CDRL1 ofSEQ ID NO:168.

In a further embodiment, the first amino acid sequence of the isolatedantigen binding protein comprises the CDRH3 of SEQ ID NO:157, the CDRH2of SEQ ID NO:144, and the CDRH1 of SEQ ID NO:130, and/or the secondamino acid sequence of the isolated antigen binding protein comprisesthe CDRL3 of SEQ ID NO:192, the CDRL2 of SEQ ID NO:177, and the CDRL1 ofSEQ ID NO:169.

In a further embodiment, the first amino acid sequence of the isolatedantigen binding protein comprises the CDRH3 of SEQ ID NO:155, the CDRH2of SEQ ID NO:135, and the CDRH1 of SEQ ID NO:127, and/or the secondamino acid sequence of the isolated antigen binding protein comprisesthe CDRL3 of SEQ ID NO:193, the CDRL2 of SEQ ID NO:178, and the CDRL1 ofSEQ ID NO:170.

In a further embodiment, the first amino acid sequence of the isolatedantigen binding protein comprises the CDRH3 of SEQ ID NO:156, the CDRH2of SEQ ID NO:142, and the CDRH1 of SEQ ID NO:129, and/or the secondamino acid sequence of the isolated antigen binding protein comprisesthe CDRL3 of SEQ ID NO:194, the CDRL2 of SEQ ID NO:173, and the CDRL1 ofSEQ ID NO:163.

In a further embodiment, the first amino acid sequence of the isolatedantigen binding protein comprises the CDRH3 of SEQ ID NO:156, the CDRH2of SEQ ID NO:141, and the CDRH1 of SEQ ID NO:128, and/or the secondamino acid sequence of the isolated antigen binding protein comprisesthe CDRL3 of SEQ ID NO:194, the CDRL2 of SEQ ID NO:173, and the CDRL1 ofSEQ ID NO:163.

In a further embodiment, the first amino acid sequence of the isolatedantigen binding protein comprises the CDRH3 of SEQ ID NO:156, the CDRH2of SEQ ID NO:143, and the CDRH1 of SEQ ID NO:128, and/or the secondamino acid sequence of the isolated antigen binding protein comprisesthe CDRL3 of SEQ ID NO:194, the CDRL2 of SEQ ID NO:179, and the CDRL1 ofSEQ ID NO:163.

In a further embodiment, the antigen binding protein comprises at leasttwo CDRH sequences of heavy chain sequences H1, H2, H3, H4, H5, H6, H7,H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 or H18, as shown in Table4A. In again a further embodiment, the antigen binding protein comprisesat least two CDRL sequences of light chain sequences L1, L2, L3, L4, L5,L6, L7, L8, L9, L10, L11, L12, L13, L14, L15, L16, L17 or L18, as shownin Table 4B. In still a further embodiment, the antigen binding proteincomprises at least two CDRH sequences of heavy chain sequences H1, H2,H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 orH18 as shown in Table 4A, and at least two CDRLs of light chainsequences L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, L13, L14,L15, L16, L17 or L18 as shown in Table 4B.

In again another embodiment, the antigen binding protein comprises theCDRH1, CDRH2, and CDRH3 sequences of heavy chain sequences H1, H2, H3,H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 or H18 asshown in Table 4A. In yet another embodiment, the antigen bindingprotein comprises the CDRL1, CDRL2, and CDRL3 sequences of light chainsequences L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, L13, L14,L15, L16, L17 or L18 as shown in Table 4B.

In yet another embodiment, the antigen binding protein comprises all sixCDRs of L1 and H1, or L2 and H2, or L3 and H3, or L3 and H4, or L4 andH5, or L5 and H6, or L6 and H7, or L7 and H8, or L8 and H7, or L9 andH9, or L9 and H10, or L10 and H11, or L11 and H11, or L12 and H12, orL13 and H13, or L14 and H14, or L15 and H15, or L16 and H16, or L17 andH17, or L18 and H18, as shown in Tables 4A and 4B.

TABLE 4A Heavy Chain Sequences Full Heavy Full Heavy Variable HeavyVariable Heavy CDRH1 CDRH2 CDRH3 Ref (H#) SEQ ID NO (VH#) SEQ ID NO SEQID NO SEQ ID NO SEQ ID NO 17C3 H1 30 V_(H)1 66 122 133 146 22H5 H2 31V_(H)2 67 122 133 147 16H7 H3 32 V_(H)3 68 122 133 148 24H11 H4 33V_(H)4 69 122 134 148 18G1 H5 34 V_(H)5 70 124 136 150 17D8 H6 35 V_(H)671 124 138 152 26H11 H7 36 V_(H)7 72 124 139 152 12E4 H8 37 V_(H)8 73124 137 151 12C11 H7 37 V_(H)8 73 124 137 151 21H2 H9 38 V_(H)9 74 131140 153 21B4 H10 39 V_(H)10 75 125 140 153 18B11.1 H11 40 V_(H)11 76 123135 149 18B11.2 H11 40 V_(H)11 77 123 135 149 20D4 H12 41 V_(H)12 78 121132 145 46D11 H13 42 V_(H)13 79 126 133 154 40D2 H14 46 V_(H)14 80 130144 157 39F7 H16 44 V_(H)16 82 129 142 156 39F11 H17 43 V_(H)17 83 128141 156 37D3 H15 47 V_(H)15 81 127 135 155 39G5 H18 45 V_(H)18 84 128143 156

TABLE 4B Light Chain Sequences Full Light Full Light Variable LightVariable Light CDRL1 CDRL2 CDRL3 Ref (L#) SEQ ID NO (VH#) SEQ ID NO SEQID NO SEQ ID NO SEQ ID NO 17C3 L1 48 V_(L)1 85 167 176 190 22H5 L2 49V_(L)2 86 167 176 189 16H7 L3 50 V_(L)3 87 166 176 188 24H11 L3 50V_(L)3 87 166 176 188 18G1 L4 51 V_(L)4 88 161 174 183 17D8 L5 52 V_(L)589 162 173 184 26H11 L6 53 V_(L)6 90 162 173 186 12E4 L7 54 V_(L)7 91164 173 186 12C11 L8 55 V_(L)8 92 160 173 182 21H2 L9 56 V_(L)9 93 163173 185 21B4 L9 56 V_(L)9 93 163 173 185 18B11.1 L10 57 V_(L)10 94 159172 181 18B11.2 L11 58 V_(L)11 95 165 175 187 20D4 L12 59 V_(L)12 96 158171 180 46D11 L13 60 V_(L)13 97 168 171 191 40D2 L14 61 V_(L)14 98 169177 192 39F7 L16 63 V_(L)16 100 163 173 194 37D3 L15 62 V_(L)15 99 170178 193 39F11 L17 64 V_(L)17 101 163 173 194 39G5 L18 65 V_(L)18 102 163179 194

In one aspect, the isolated antigen binding proteins that specificallybind (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) acomplex comprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4provided herein can be a monoclonal antibody, a polyclonal antibody, arecombinant antibody, a human antibody, a humanized antibody, a chimericantibody, a multispecific antibody, or an antibody fragment thereof.

In another embodiment, the antibody fragment of the isolatedantigen-binding proteins provided herein can be a Fab fragment, a Fab′fragment, an F(ab′)₂ fragment, an Fv fragment, a diabody, or a singlechain antibody molecule.

In a further embodiment, an isolated antigen binding protein thatspecifically (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or(iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c,and FGFR4 provided herein is a human antibody and can be of the IgG1-,IgG2-IgG3- or IgG4-type.

In another embodiment, an isolated antigen binding protein thatspecifically binds (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4;or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c,FGFR3c, and FGFR4 comprises a light or a heavy chain polypeptide as setforth in Tables 1A-1B. In some embodiments, an antigen binding proteinthat specifically binds (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c orFGFR4; or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c,FGFR3c, and FGFR4 comprises a variable light or variable heavy domainsuch as those listed in Tables 2A-2B. In still other embodiments, anantigen binding protein that specifically binds (i) β-Klotho; (ii)FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klothoand one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 comprises one, two or threeCDRHs or one, two or three CDRLs as set forth in Tables 3A-3B, 4A-4B andTable 6C, infra. Such antigen binding proteins, and indeed any of theantigen binding proteins disclosed herein, can be PEGylated with one ormore PEG molecules, for examples PEG molecules having a molecular weightselected from the group consisting of 5K, 10K, 20K, 40K, 50K, 60K, 80K,100K or greater than 100K.

In yet another aspect, any antigen binding protein that specificallybinds (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) acomplex comprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4provided herein can be coupled to a labeling group and can compete forbinding to the extracellular portion of (i) β-Klotho; (ii) FGFR1c,FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and oneof FGFR1c, FGFR2c, FGFR3c, and FGFR4 with an antigen binding protein ofone of the isolated antigen binding proteins provided herein. In oneembodiment, the isolated antigen binding protein provided herein canreduce blood glucose levels, decrease triglyceride and cholesterollevels or improve other glycemic parameters and cardiovascular riskfactors when administered to a patient.

As will be appreciated, for any antigen binding protein comprising morethan one CDR provided in Tables 3A-3B, and 4A-4B, any combination ofCDRs independently selected from the depicted sequences may be useful.Thus, antigen binding proteins with one, two, three, four, five or sixof independently selected CDRs can be generated. However, as will beappreciated by those in the art, specific embodiments generally utilizecombinations of CDRs that are non-repetitive, e.g., antigen bindingproteins are generally not made with two CDRH2 regions, etc.

Some of the antigen binding proteins that specifically bind (i)β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 thatare provided herein are discussed in more detail below.

Antigen Binding Proteins and Binding Epitopes and Binding Domains

When an antigen binding protein is said to bind an epitope on (i)β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4, or theextracellular domain of β-Klotho, FGFR1c, FGFR2c, FGFR3c or FGFR4, forexample, what is meant is that the antigen binding protein specificallybinds to a specified portion of (i) β-Klotho; (ii) FGFR1c, FGFR2c,FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one ofFGFR1c, FGFR2c, FGFR3c, and FGFR4. In some embodiments, e.g., in certaincases where the antigen binding protein binds only FGFR1c or β-Klotho,the antigen binding protein can specifically bind to a polypeptideconsisting of specified residues (e.g., a specified segment of β-Klotho,FGFR1c, FGFR2c, FGFR3c or FGFR4, such as those residues disclosed inExample 14). In other embodiments, e.g., in certain cases where anantigen binding protein interacts with both β-Klotho and one or more ofFGFR1c, FGFR2c, FGFR3c and FGFR4, the antigen binding protein can bindresidues, sequences of residues, or regions in both β-Klotho and FGFR1c,FGFR2c, FGFR3c or FGFR4, depending on which receptor the antigen bindingprotein recognizes. In still other embodiments the antigen bindingprotein will bind residues, sequence or residues or regions of a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4, forexample FGFR1c.

In any of the foregoing embodiments, such an antigen binding proteindoes not need to contact every residue of (i) β-Klotho; (ii) FGFR1c,FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and oneof FGFR1c, FGFR2c, FGFR3c, and FGFR4, or the extracellular domain of therecited proteins or complexes. Nor does every single amino acidsubstitution or deletion within (i) β-Klotho; (ii) FGFR1c, FGFR2c,FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one ofFGFR1c, FGFR2c, FGFR3c, and FGFR4, or the extracellular domain of therecited proteins or complexes, necessarily significantly affect bindingaffinity.

Epitope specificity and the binding domain(s) of an antigen bindingprotein can be determined by a variety of methods. Some methods, forexample, can use truncated portions of an antigen. Other methods utilizeantigen mutated at one or more specific residues, such as by employingan alanine scanning or arginine scanning-type approach or by thegeneration and study of chimeric proteins in which various domains,regions or amino acids are swapped between two proteins (e.g., mouse andhuman forms of one or more of the antigens or target proteins), or byprotease protection assays.

Competing Antigen Binding Proteins

In another aspect, antigen binding proteins are provided that competewith one of the exemplified antibodies or functional fragments forbinding to (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii)a complex comprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, andFGFR4. Such antigen binding proteins can also bind to the same epitopeas one of the herein exemplified antigen binding proteins, or anoverlapping epitope. Antigen binding proteins and fragments that competewith or bind to the same epitope as the exemplified antigen bindingproteins are expected to show similar functional properties. Theexemplified antigen binding proteins and fragments include those withthe heavy and light chains H1-H18 and L1-L18, variable region domainsV_(L)1-V_(L)18 and V_(H)1-V_(H)18, and CDRs provided herein, includingthose in Tables 1, 2, 3, and 4. Thus, as a specific example, the antigenbinding proteins that are provided include those that compete with anantibody comprising:

(a) 1, 2, 3, 4, 5 or all 6 of the CDRs listed for an antibody listed inTables 3A and 3B, and 4A and 4B and Table 6C, infra;

(b) a V_(H) and a V_(L) selected from V_(L)1-V_(L)18 and V_(H)1-V_(H)18and listed for an antibody listed in Tables 2A and 2B; or

(c) two light chains and two heavy chains as specified for an antibodylisted in Tables 1A and 12B and Table 6A, infra.

Thus, in one embodiment, the present disclosure provides antigen bindingproteins that competes for binding to (i) β-Klotho; (ii) FGFR1c, FGFR2c,FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one ofFGFR1c, FGFR2c, FGFR3c, and FGFR4 with a reference antibody, wherein thereference antibody comprises a combination of light chain and heavychain variable domain sequences selected from the group consisting ofL1H1, L2H2, L3H3, L3H4, L4H5, L5H6, L6H7, L7H8, L8H8, L9H9, L9H10,L10H11, L11H11, L12H12, L13H13, L14H14, L15H15, L16H16, L17H17 orL18H18. In another embodiment, the present disclosure provides humanantibodies that compete for binding to (i) β-Klotho; (ii) FGFR1c,FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and oneof FGFR1c, FGFR2c, FGFR3c, and FGFR4 with a reference antibody, whereinthe reference antibody is 17C3, 22H5, 16H7, 24H11, 18G1, 17D8, 26H11,12E4, 12C11, 21H2, 21B4, 18B11.1, 18B11.2, 20D4, 46D11, 40D2, 37D3,39F7, 39F1 or 39G5.

In a further embodiment, an isolated human antibody is provided thatbinds to (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) acomplex comprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4with substantially the same Kd as a reference antibody; initiatesFGF21-like signaling in an in vitro ELK-Luciferase assay to the samedegree as a reference antibody; lowers blood glucose; lowers serum lipidlevels; and/or competes for binding with said reference antibody to (i)β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4,wherein the reference antibody is selected from the group consisting of17C3, 22H5, 16H7, 24H11, 18G1, 17D8, 26H11, 12E4, 12C11, 21H2, 21B4,18B11.1, 18B11.2, 20D4, 46D11, 40D2, 37D3, 39F7, 39F1 or 39G5.

The ability to compete with an antibody can be determined using anysuitable assay, such as that described in Example 8, in which antigenbinding proteins 17C3, 22H5, 16H7, 24H11, 18G1, 17D8, 26H11, 12E4,12C11, 21H2, 21B4, 18B11.1, 18B11.2, 20D4, 46D11, 40D2, 37D3, 39F7, 39F1or 39G5 can be used as the reference antibody.

Monoclonal Antibodies

The antigen binding proteins that are provided include monoclonalantibodies that bind to (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c orFGFR4; or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c,FGFR3c, and FGFR4, and induce FGF21-like signaling to various degrees.Monoclonal antibodies can be produced using any technique known in theart, e.g., by immortalizing spleen cells harvested from the transgenicanimal after completion of the immunization schedule. The spleen cellscan be immortalized using any technique known in the art, e.g., byfusing them with myeloma cells to produce hybridomas. Myeloma cells foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render them incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas). Examples of suitable cell lines for use in mouse fusionsinclude Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO,NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XXO Bul; examples of celllines used in rat fusions include R210.RCY3, Y3-Ag 1.2.3, IR983F and4B210. Other cell lines useful for cell fusions are U-266, GM1500-GRG2,LICR-LON-HMy2 and UC729-6.

In some instances, a hybridoma cell line is produced by immunizing ananimal (e.g., a transgenic animal having human immunoglobulin sequences)with a FGFR1c, β-Klotho or FGFR1c and/or β-Klotho immunogen (e.g., asoluble complex comprising the extracellular domains of FGFR1c, FGFR2c,FGFR3c or FGFR4 and/or β-Klotho as shown in Examples 2, and 3; membraneson which the extracellular domains of FGFR1c, FGFR2c, FGFR3c or FGFR4and/or β-Klotho are expressed, as shown in Examples 1 and 3; or wholecells expressing FGFR1c and/or β-Klotho, as shown in Examples 1 and 3);harvesting spleen cells from the immunized animal; fusing the harvestedspleen cells to a myeloma cell line, thereby generating hybridoma cells;establishing hybridoma cell lines from the hybridoma cells, andidentifying a hybridoma cell line that produces an antibody that bindsto (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) acomplex comprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4(e.g., as described in the Example 4) and can induce FGF21-likesignaling (e.g., as described in Examples 5-7). Such hybridoma celllines, and the monoclonal antibodies produced by them, form aspects ofthe present disclosure.

Monoclonal antibodies secreted by a hybridoma cell line can be purifiedusing any technique known in the art. Hybridomas or mAbs can be furtherscreened to identify mAbs with particular properties, such as theability to induce FGF21-like signaling. Examples of such screens areprovided herein.

Chimeric and Humanized Antibodies

Chimeric and humanized antibodies based upon the foregoing sequences canreadily be generated. One example is a chimeric antibody, which is anantibody composed of protein segments from different antibodies that arecovalently joined to produce functional immunoglobulin light or heavychains or immunologically functional portions thereof. Generally, aportion of the heavy chain and/or light chain is identical with orhomologous to a corresponding sequence in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is/are identical with orhomologous to a corresponding sequence in antibodies derived fromanother species or belonging to another antibody class or subclass. Formethods relating to chimeric antibodies, see, for example, U.S. Pat. No.4,816,567; and Morrison et al., 1985, Proc. Natl. Acad. Sci. USA81:6851-6855, which are hereby incorporated by reference. CDR graftingis described, for example, in U.S. Pat. Nos. 6,180,370, 5,693,762,5,693,761, 5,585,089, and 5,530,101.

Generally, the goal of making a chimeric antibody is to create a chimerain which the number of amino acids from the intended patient/recipientspecies is maximized. One example is the “CDR-grafted” antibody, inwhich the antibody comprises one or more complementarity determiningregions (CDRs) from a particular species or belonging to a particularantibody class or subclass, while the remainder of the antibody chain(s)is/are identical with or homologous to a corresponding sequence inantibodies derived from another species or belonging to another antibodyclass or subclass. For use in humans, the variable region or selectedCDRs from a rodent antibody often are grafted into a human antibody,replacing the naturally-occurring variable regions or CDRs of the humanantibody.

One useful type of chimeric antibody is a “humanized” antibody.Generally, a humanized antibody is produced from a monoclonal antibodyraised initially in a non-human animal. Certain amino acid residues inthis monoclonal antibody, typically from non-antigen recognizingportions of the antibody, are modified to be homologous to correspondingresidues in a human antibody of corresponding isotype. Humanization canbe performed, for example, using various methods by substituting atleast a portion of a rodent variable region for the correspondingregions of a human antibody (see, e.g., U.S. Pat. Nos. 5,585,089, and5,693,762; Jones et al., 1986, Nature 321:522-525; Riechmann et al.,1988, Nature 332:323-27; Verhoeyen et al., 1988, Science 239:1534-1536).

In one aspect, the CDRs of the light and heavy chain variable regions ofthe antibodies provided herein (e.g., in Tables 3 and 4) are grafted toframework regions (FRs) from antibodies from the same, or a different,phylogenetic species. For example, the CDRs of the heavy and light chainvariable regions V_(H)1, V_(H)2, V_(H)3, V_(H)4, V_(H)5, V_(H)6, V_(H)7,V_(H)8, V_(H)9, V_(H)10, V_(H)11, V_(H)12, V_(H)13, V_(H)14, V_(H)15,V_(H)16, V_(H)17 or V_(H)18 and/or V_(L)1, V_(L)2, V_(L)3, V_(L)4,V_(L)5, V_(L)6, V_(L)7, V_(L)8, V_(L)9, V_(L)10, V_(L),11, V_(H)12,V_(L)13, V_(L)14, V_(L)15, V_(L)16, V_(L)17 or V_(L)18 can be grafted toconsensus human FRs. To create consensus human FRs, FRs from severalhuman heavy chain or light chain amino acid sequences can be aligned toidentify a consensus amino acid sequence. In other embodiments, the FRsof a heavy chain or light chain disclosed herein are replaced with theFRs from a different heavy chain or light chain. In one aspect, rareamino acids in the FRs of the heavy and light chains of an antigenbinding protein (e.g., an antibody) that specifically binds (i)β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 are notreplaced, while the rest of the FR amino acids are replaced. A “rareamino acid” is a specific amino acid that is in a position in which thisparticular amino acid is not usually found in an FR. Alternatively, thegrafted variable regions from the one heavy or light chain can be usedwith a constant region that is different from the constant region ofthat particular heavy or light chain as disclosed herein. In otherembodiments, the grafted variable regions are part of a single chain Fvantibody.

In certain embodiments, constant regions from species other than humancan be used along with the human variable region(s) to produce hybridantibodies.

Fully Human Antibodies

Fully human antibodies are also provided by the instant disclosure.Methods are available for making fully human antibodies specific for agiven antigen without exposing human beings to the antigen (“fully humanantibodies”). One specific means provided for implementing theproduction of fully human antibodies is the “humanization” of the mousehumoral immune system. Introduction of human immunoglobulin (Ig) lociinto mice in which the endogenous Ig genes have been inactivated is onemeans of producing fully human monoclonal antibodies (mAbs) in mouse, ananimal that can be immunized with any desirable antigen. Using fullyhuman antibodies can minimize the immunogenic and allergic responsesthat can sometimes be caused by administering mouse or mouse-derivedmAbs to humans as therapeutic agents.

Fully human antibodies can be produced by immunizing transgenic animals(typically mice) that are capable of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production.Antigens for this purpose typically have six or more contiguous aminoacids, and optionally are conjugated to a carrier, such as a hapten.See, e.g., Jakobovits et al., (1993) Proc. Natl. Acad. Sci. USA90:2551-2555; Jakobovits et al., (1993) Nature 362:255-258; andBruggermann et al., (1993) Year in Immunol. 7:33. In one example of sucha method, transgenic animals are produced by incapacitating theendogenous mouse immunoglobulin loci encoding the mouse heavy and lightimmunoglobulin chains therein, and inserting into the mouse genome largefragments of human genome DNA containing loci that encode human heavyand light chain proteins. Partially modified animals, which have lessthan the full complement of human immunoglobulin loci, are thencross-bred to obtain an animal having all of the desired immune systemmodifications. When administered an immunogen, these transgenic animalsproduce antibodies that are immunospecific for the immunogen but havehuman rather than murine amino acid sequences, including the variableregions. For further details of such methods, see, e.g., WO96/33735 andWO94/02602. Additional methods relating to transgenic mice for makinghuman antibodies are described in U.S. Pat. Nos. 5,545,807; 6,713,610;6,673,986; 6,162,963; 5,545,807; 6,300,129; 6,255,458; 5,877,397;5,874,299 and 5,545,806; in PCT publications WO91/10741, WO90/04036, andin EP 546073B1 and EP 546073A1.

The transgenic mice described above, referred to herein as “HuMab” mice,contain a human immunoglobulin gene minilocus that encodes unrearrangedhuman heavy ([μ, mu] and [γ, gamma]) and [κ, kappa] light chainimmunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ [mu] and κ [kappa] chain loci (Lonberg etal., 1994, Nature 368:856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or [κ, kappa] and in response to immunization,and the introduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgG [κ,kappa] monoclonal antibodies (Lonberg et al., supra.; Lonberg andHuszar, (1995) Intern. Rev. Immunol. 13: 65-93; Harding and Lonberg,(1995) Ann. N.Y. Acad. Sci. 764:536-546). The preparation of HuMab miceis described in detail in Taylor et al., (1992) Nucleic Acids Research20:6287-6295; Chen et al., (1993) International Immunology 5:647-656;Tuaillon et al., (1994) J. Immunol. 152:2912-2920; Lonberg et al.,(1994) Nature 368:856-859; Lonberg, (1994) Handbook of Exp. Pharmacology113:49-101; Taylor et al., (1994) International Immunology 6:579-591;Lonberg and Huszar, (1995) Intern. Rev. Immunol. 13:65-93; Harding andLonberg, (1995) Ann. N.Y. Acad. Sci. 764:536-546; Fishwild et al.,(1996) Nature Biotechnology 14:845-851; the foregoing references arehereby incorporated by reference in their entirety for all purposes.See, further U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; aswell as U.S. Pat. No. 5,545,807; International Publication Nos. WO93/1227; WO 92/22646; and WO 92/03918, the disclosures of all of whichare hereby incorporated by reference in their entirety for all purposes.Technologies utilized for producing human antibodies in these transgenicmice are disclosed also in WO 98/24893, and Mendez et al., (1997) NatureGenetics 15:146-156, which are hereby incorporated by reference. Forexample, the HCo7 and HCo12 transgenic mice strains can be used togenerate antigen binding proteins (e.g., antibodies) that bind to (i)β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 and mayinduce FGF21-like signaling. Further details regarding the production ofhuman antibodies using transgenic mice are provided in the examplesbelow.

Using hybridoma technology, antigen-specific human mAbs with the desiredspecificity can be produced and selected from the transgenic mice suchas those described above. Such antibodies can be cloned and expressedusing a suitable vector and host cell, or the antibodies can beharvested from cultured hybridoma cells.

Fully human antibodies can also be derived from phage-display libraries(as disclosed in Hoogenboom et al., (1991) J. Mol. Biol. 227:381; andMarks et al., (1991) J. Mol. Biol. 222:581). Phage display techniquesmimic immune selection through the display of antibody repertoires onthe surface of filamentous bacteriophage, and subsequent selection ofphage by their binding to an antigen of choice. One such technique isdescribed in PCT Publication No. WO 99/10494 (hereby incorporated byreference), which describes the isolation of high affinity andfunctional agonistic antibodies for MPL- and msk-receptors using such anapproach.

Bispecific or Bifunctional Antigen Binding Proteins

Also provided are bispecific and bifunctional antibodies that includeone or more CDRs or one or more variable regions as described above. Abispecific or bifunctional antibody in some instances can be anartificial hybrid antibody having two different heavy/light chain pairsand two different binding sites. Bispecific antibodies can be producedby a variety of methods including, but not limited to, fusion ofhybridomas or linking of Fab′ fragments. See, e.g., Songsivilai andLachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al., 1992,J. Immunol. 148:1547-1553. When an antigen binding protein of theinstant disclosure binds (i) both β-Klotho and one or more of FGFR1c,FGFR2c, FGFR3c or FGFR4; or (ii) a complex comprising β-Klotho and oneof FGFR1c, FGFR2c, FGFR3c, and FGFR4, the binding may lead to theactivation of FGF21-like activity as measured by the FGF21-likefunctional and signaling assays described in Examples 5-7; when such anantigen binding protein is an antibody it is referred to as an agonisticantibody.

Various Other Forms

Some of the antigen binding proteins that specifically bind (i)β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 thatare provided in the present disclosure include variant forms of theantigen binding proteins disclosed herein (e.g., those having thesequences listed in Tables 1-4).

In various embodiments, the antigen binding proteins disclosed hereincan comprise one or more non-naturally occurring amino acids. Forinstance, some of the antigen binding proteins have one or morenon-naturally occurring amino acid substitutions in one or more of theheavy or light chains, variable regions or CDRs listed in Tables 1-4.Examples of non-naturally amino acids (which can be substituted for anynaturally-occurring amino acid found in any sequence disclosed herein,as desired) include: 4-hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxyl-terminal direction, in accordance withstandard usage and convention. A non-limiting lists of examples ofnon-naturally occurring amino acids that can be inserted into an antigenbinding protein sequence or substituted for a wild-type residue in anantigen binding sequence include β-amino acids, homoamino acids, cyclicamino acids and amino acids with derivatized side chains. Examplesinclude (in the L-form or D-form; abbreviated as in parentheses):citrulline (Cit), homocitrulline (hCit), Nα-methylcitrulline (NMeCit),Nα-methylhomocitrulline (Nα-MeHoCit), ornithine (Orn),Nα-Methylornithine (Nα-MeOrn or NMeOrn), sarcosine (Sar), homolysine(hLys or hK), homoarginine (hArg or hR), homoglutamine (hQ),Nα-methylarginine (NMeR), Nα-methylleucine (Nα-MeL or NMeL),N-methylhomolysine (NMeHoK), Nα-methylglutamine (NMeQ), norleucine(Nle), norvaline (Nva), 1,2,3,4-tetrahydroisoquinoline (Tic),Octahydroindole-2-carboxylic acid (Oic), 3-(1-naphthyl)alanine (1-Nal),3-(2-naphthyl)alanine (2-Nal), 1,2,3,4-tetrahydroisoquinoline (Tic),2-indanylglycine (IgI), para-iodophenylalanine (pI-Phe),para-aminophenylalanine (4AmP or 4-Amino-Phe), 4-guanidino phenylalanine(Guf), glycyllysine (abbreviated “K(Nε-glycyl)” or “K(glycyl)” or“K(gly)”), nitrophenylalanine (nitrophe), aminophenylalanine (aminopheor Amino-Phe), benzylphenylalanine (benzylphe), γ-carboxyglutamic acid(γ-carboxyglu), hydroxyproline (hydroxypro), p-carboxyl-phenylalanine(Cpa), α-aminoadipic acid (Aad), Nα-methyl valine (NMeVal), N-α-methylleucine (NMeLeu), Nα-methylnorleucine (NMeNle), cyclopentylglycine(Cpg), cyclohexylglycine (Chg), acetylarginine (acetylarg), α,β-diaminopropionoic acid (Dpr), α, γ-diaminobutyric acid (Dab),diaminopropionic acid (Dap), cyclohexylalanine (Cha),4-methyl-phenylalanine (MePhe), β, β-diphenyl-alanine (BiPhA),aminobutyric acid (Abu), 4-phenyl-phenylalanine (or biphenylalanine;4Bip), α-amino-isobutyric acid (Aib), beta-alanine, beta-aminopropionicacid, piperidinic acid, aminocaprioic acid, aminoheptanoic acid,aminopimelic acid, desmosine, diaminopimelic acid, N-ethylglycine,N-ethylaspargine, hydroxylysine, allo-hydroxylysine, isodesmosine,allo-isoleucine, N-methylglycine, N-methylisoleucine, N-methylvaline,4-hydroxyproline (Hyp), γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, ω-methylarginine, 4-Amino-O-PhthalicAcid (4APA), and other similar amino acids, and derivatized forms of anyof those specifically listed.

Additionally, the antigen binding proteins can have one or moreconservative amino acid substitutions in one or more of the heavy orlight chains, variable regions or CDRs listed in Tables 1-4.Naturally-occurring amino acids can be divided into classes based oncommon side chain properties:

1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

3) acidic: Asp, Glu;

4) basic: His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

Conservative amino acid substitutions can involve exchange of a memberof one of these classes with another member of the same class.Conservative amino acid substitutions can encompass non-naturallyoccurring amino acid residues, which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. See Table 5, infra. These include peptidomimetics and otherreversed or inverted forms of amino acid moieties.

Non-conservative substitutions can involve the exchange of a member ofone of the above classes for a member from another class. Suchsubstituted residues can be introduced into regions of the antibody thatare homologous with human antibodies, or into the non-homologous regionsof the molecule.

In making such changes, according to certain embodiments, thehydropathic index of amino acids can be considered. The hydropathicprofile of a protein is calculated by assigning each amino acid anumerical value (“hydropathy index”) and then repetitively averagingthese values along the peptide chain. Each amino acid has been assigneda hydropathic index on the basis of its hydrophobicity and chargecharacteristics. They are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic profile in conferring interactivebiological function on a protein is understood in the art (see, e.g.,Kyte et al., 1982, J. Mol. Biol. 157:105-131). It is known that certainamino acids can be substituted for other amino acids having a similarhydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, in certainembodiments, the substitution of amino acids whose hydropathic indicesare within ±2 is included. In some aspects, those which are within ±1are included, and in other aspects, those within ±0.5 are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments, as inthe present case. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigen-binding or immunogenicity, that is, with a biological propertyof the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within ±2 is included, in otherembodiments, those which are within ±1 are included, and in still otherembodiments, those within ±0.5 are included. In some instances, one canalso identify epitopes from primary amino acid sequences on the basis ofhydrophilicity. These regions are also referred to as “epitopic coreregions.”

Exemplary conservative amino acid substitutions are set forth in Table5.

TABLE 5 Conservative Amino Acid Substitutions Original Residue ExemplarySubstitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn GluAsp Gly Pro His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu MetLeu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile,Leu

A skilled artisan will be able to determine suitable variants ofpolypeptides as set forth herein using well-known techniques coupledwith the information provided herein. One skilled in the art canidentify suitable areas of the molecule that can be changed withoutdestroying activity by targeting regions not believed to be importantfor activity. The skilled artisan also will be able to identify residuesand portions of the molecules that are conserved among similarpolypeptides. In further embodiments, even areas that can be importantfor biological activity or for structure can be subject to conservativeamino acid substitutions without destroying the biological activity orwithout adversely affecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in a protein that correspond toamino acid residues important for activity or structure in similarproteins. One skilled in the art can opt for chemically similar aminoacid substitutions for such predicted important amino acid residues.

One skilled in the art can also analyze the 3-dimensional structure andamino acid sequence in relation to that structure in similarpolypeptides. In view of such information, one skilled in the art canpredict the alignment of amino acid residues of an antibody with respectto its three dimensional structure. One skilled in the art can choosenot to make radical changes to amino acid residues predicted to be onthe surface of the protein, since such residues can be involved inimportant interactions with other molecules. Moreover, one skilled inthe art can generate test variants containing a single amino acidsubstitution at each desired amino acid residue. These variants can thenbe screened using assays for FGF21-like signaling, (see the Examplesprovided herein) thus yielding information regarding which amino acidscan be changed and which must not be changed. In other words, based oninformation gathered from such routine experiments, one skilled in theart can readily determine the amino acid positions where furthersubstitutions should be avoided either alone or in combination withother mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See, Moult, (1996) Curr. Op. in Biotech.7:422-427; Chou et al., (1974) Biochem. 13:222-245; Chou et al., (1974)Biochemistry 113:211-222; Chou et al., (1978) Adv. Enzymol. Relat. AreasMol. Biol. 47:45-148; Chou et al., (1979) Ann. Rev. Biochem. 47:251-276;and Chou et al., (1979) Biophys. J. 26:367-384. Moreover, computerprograms are currently available to assist with predicting secondarystructure. One method of predicting secondary structure is based uponhomology modeling. For example, two polypeptides or proteins that have asequence identity of greater than 30%, or similarity greater than 40%can have similar structural topologies. The growth of the proteinstructural database (PDB) has provided enhanced predictability ofsecondary structure, including the potential number of folds within apolypeptide's or protein's structure. See, Holm et al., (1999) Nucl.Acid. Res. 27:244-247. It has been suggested (Brenner et al., (1997)Curr. Op. Struct. Biol. 7:369-376) that there are a limited number offolds in a given polypeptide or protein and that once a critical numberof structures have been resolved, structural prediction will becomedramatically more accurate.

Additional methods of predicting secondary structure include “threading”(Jones, (1997) Curr. Opin. Struct. Biol. 7:377-387; Sippl et al., (1996)Structure 4:15-19), “profile analysis” (Bowie et al., (1991) Science253:164-170; Gribskov et al., (1990) Meth. Enzym. 183:146-159; Gribskovet al., (1987) Proc. Nat. Acad. Sci. 84:4355-4358), and “evolutionarylinkage” (See, Holm, (1999) supra; and Brenner, (1997) supra).

In some embodiments, amino acid substitutions are made that: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterligand or antigen binding affinities, and/or (4) confer or modify otherphysicochemical or functional properties on such polypeptides. Forexample, single or multiple amino acid substitutions (in certainembodiments, conservative amino acid substitutions) can be made in thenaturally-occurring sequence. Substitutions can be made in that portionof the antibody that lies outside the domain(s) forming intermolecularcontacts). In such embodiments, conservative amino acid substitutionscan be used that do not substantially change the structuralcharacteristics of the parent sequence (e.g., one or more replacementamino acids that do not disrupt the secondary structure thatcharacterizes the parent or native antigen binding protein). Examples ofart-recognized polypeptide secondary and tertiary structures aredescribed in Proteins, Structures and Molecular Principles (Creighton,Ed.), 1984, W. H. New York: Freeman and Company; Introduction to ProteinStructure (Branden and Tooze, eds.), 1991, New York: Garland Publishing;and Thornton et al., (1991) Nature 354:105, which are each incorporatedherein by reference.

Additional preferred antibody variants include cysteine variants whereinone or more cysteine residues in the parent or native amino acidsequence are deleted from or substituted with another amino acid (e.g.,serine). Cysteine variants are useful, inter alia when antibodies mustbe refolded into a biologically active conformation. Cysteine variantscan have fewer cysteine residues than the native antibody, and typicallyhave an even number to minimize interactions resulting from unpairedcysteines.

The heavy and light chains, variable regions domains and CDRs that aredisclosed can be used to prepare polypeptides that contain an antigenbinding region that can specifically bind (i) β-Klotho; (ii) FGFR1c,FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and oneof FGFR1c, FGFR2c, FGFR3c, and FGFR4 and may induce FGF21-likesignaling. For example, one or more of the CDRs listed in Tables 3 and 4can be incorporated into a molecule (e.g., a polypeptide) covalently ornoncovalently to make an immunoadhesion. An immunoadhesion canincorporate the CDR(s) as part of a larger polypeptide chain, cancovalently link the CDR(s) to another polypeptide chain, or canincorporate the CDR(s) noncovalently. The CDR(s) enable theimmunoadhesion to bind specifically to a particular antigen of interest(e.g., (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) acomplex comprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4or an epitope thereon).

The heavy and light chains, variable regions domains and CDRs that aredisclosed can be used to prepare polypeptides that contain an antigenbinding region that can specifically bind (i) β-Klotho; (ii) FGFR1c,FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and oneof FGFR1c, FGFR2c, FGFR3c, and FGFR4 and may induce FGF21-likesignaling. For example, one or more of the CDRs listed in Tables 3 and 4can be incorporated into a molecule (e.g., a polypeptide) that isstructurally similar to a “half” antibody comprising the heavy chain,the light chain of an antigen binding protein paired with a Fc fragmentso that the antigen binding region is monovalent (like a Fab fragment)but with a dimeric Fc moiety.

Mimetics (e.g., “peptide mimetics” or “peptidomimetics”) based upon thevariable region domains and CDRs that are described herein are alsoprovided. These analogs can be peptides, non-peptides or combinations ofpeptide and non-peptide regions. Fauchere, 1986, Adv. Drug Res. 15:29;Veber and Freidinger, 1985, TINS p. 392; and Evans et al., 1987, J. Med.Chem. 30:1229, which are incorporated herein by reference for anypurpose. Peptide mimetics that are structurally similar totherapeutically useful peptides can be used to produce a similartherapeutic or prophylactic effect. Such compounds are often developedwith the aid of computerized molecular modeling. Generally,peptidomimetics are proteins that are structurally similar to anantibody displaying a desired biological activity, such as here theability to specifically bind (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3cor FGFR4; or (iii) a complex comprising β-Klotho and one of FGFR1c,FGFR2c, FGFR3c, and FGFR4, but have one or more peptide linkagesoptionally replaced by a linkage selected from: —CH₂NH—, —CH₂S—,—CH₂—CH₂—, —CH—CH-(cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, bymethods well known in the art. Systematic substitution of one or moreamino acids of a consensus sequence with a D-amino acid of the same type(e.g., D-lysine in place of L-lysine) can be used in certain embodimentsto generate more stable proteins. In addition, constrained peptidescomprising a consensus sequence or a substantially identical consensussequence variation can be generated by methods known in the art (Rizoand Gierasch, 1992, Ann. Rev. Biochem. 61:387), incorporated herein byreference), for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

Derivatives of the antigen binding proteins that specifically bind (i)β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 thatare described herein are also provided. The derivatized antigen bindingproteins can comprise any molecule or substance that imparts a desiredproperty to the antibody or fragment, such as increased half-life in aparticular use. The derivatized antigen binding protein can comprise,for example, a detectable (or labeling) moiety (e.g., a radioactive,colorimetric, antigenic or enzymatic molecule, a detectable bead (suchas a magnetic or electrodense (e.g., gold) bead), or a molecule thatbinds to another molecule (e.g., biotin or streptavidin)), a therapeuticor diagnostic moiety (e.g., a radioactive, cytotoxic, orpharmaceutically active moiety), or a molecule that increases thesuitability of the antigen binding protein for a particular use (e.g.,administration to a subject, such as a human subject, or other in vivoor in vitro uses). Examples of molecules that can be used to derivatizean antigen binding protein include albumin (e.g., human serum albumin)and polyethylene glycol (PEG). Albumin-linked and PEGylated derivativesof antigen binding proteins can be prepared using techniques well knownin the art. Certain antigen binding proteins include a PEGylated singlechain polypeptide as described herein. In one embodiment, the antigenbinding protein is conjugated or otherwise linked to transthyretin (TTR)or a TTR variant. The TTR or TTR variant can be chemically modifiedwith, for example, a chemical selected from the group consisting ofdextran, poly(n-vinyl pyrrolidone), polyethylene glycols, propropyleneglycol homopolymers, polypropylene oxide/ethylene oxide co-polymers,polyoxyethylated polyols and polyvinyl alcohols.

Other derivatives include covalent or aggregative conjugates of theantigen binding proteins that specifically bind (i) β-Klotho; (ii)FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klothoand one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 that are disclosed hereinwith other proteins or polypeptides, such as by expression ofrecombinant fusion proteins comprising heterologous polypeptides fusedto the N-terminus or C-terminus of an antigen binding protein thatinduces FGF21-like signaling. For example, the conjugated peptide can bea heterologous signal (or leader) polypeptide, e.g., the yeastalpha-factor leader, or a peptide such as an epitope tag. An antigenbinding protein-containing fusion protein of the present disclosure cancomprise peptides added to facilitate purification or identification ofan antigen binding protein that specifically binds (i) β-Klotho; (ii)FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klothoand one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 (e.g., a poly-His tag) andthat can induce FGF21-like signaling. An antigen binding protein thatspecifically binds (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4;or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c,FGFR3c, and FGFR4 also can be linked to the FLAG peptide as described inHopp et al., 1988, Bio/Technology 6:1204; and U.S. Pat. No. 5,011,912.The FLAG peptide is highly antigenic and provides an epitope reversiblybound by a specific monoclonal antibody (mAb), enabling rapid assay andfacile purification of expressed recombinant protein. Reagents usefulfor preparing fusion proteins in which the FLAG peptide is fused to agiven polypeptide are commercially available (Sigma, St. Louis, Mo.).

Multimers that comprise one or more antigen binding proteins thatspecifically bind (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or(iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c,and FGFR4 form another aspect of the present disclosure. Multimers cantake the form of covalently-linked or non-covalently-linked dimers,trimers, or higher multimers. Multimers comprising two or more antigenbinding proteins that bind (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c orFGFR4; or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c,FGFR3c, and FGFR4 and which may induce FGF21-like signaling arecontemplated for use as therapeutics, diagnostics and for other uses aswell, with one example of such a multimer being a homodimer. Otherexemplary multimers include heterodimers, homotrimers, heterotrimers,homotetramers, heterotetramers, etc.

One embodiment is directed to multimers comprising multiple antigenbinding proteins that specifically bind (i) β-Klotho; (ii) FGFR1c,FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and oneof FGFR1c, FGFR2c, FGFR3c, and FGFR4 joined via covalent or non-covalentinteractions between peptide moieties fused to an antigen bindingprotein that specifically binds (i) β-Klotho; (ii) FGFR1c, FGFR2c,FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one ofFGFR1c, FGFR2c, FGFR3c, and FGFR4. Such peptides can be peptide linkers(spacers), or peptides that have the property of promotingmultimerization. Leucine zippers and certain polypeptides derived fromantibodies are among the peptides that can promote multimerization ofantigen binding proteins attached thereto, as described in more detailherein.

In particular embodiments, the multimers comprise from two to fourantigen binding proteins that bind (i) β-Klotho; (ii) FGFR1c, FGFR2c,FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one ofFGFR1c, FGFR2c, FGFR3c, and FGFR4. The antigen binding protein moietiesof the multimer can be in any of the forms described above, e.g.,variants or fragments. Preferably, the multimers comprise antigenbinding proteins that have the ability to specifically bind (i)β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4.

In one embodiment, an oligomer is prepared using polypeptides derivedfrom immunoglobulins. Preparation of fusion proteins comprising certainheterologous polypeptides fused to various portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al., (1991) Proc. Natl. Acad. Sci. USA 88:10535; Byrn etal., (1990) Nature 344:677; and Hollenbaugh et al., 1992 “Constructionof Immunoglobulin Fusion Proteins”, in Current Protocols in Immunology,Suppl. 4, pages 10.19.1-10.19.11.

One embodiment comprises a dimer comprising two fusion proteins createdby fusing an antigen binding protein that specifically binds (i)β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 to theFc region of an antibody. The dimer can be made by, for example,inserting a gene fusion encoding the fusion protein into an appropriateexpression vector, expressing the gene fusion in host cells transformedwith the recombinant expression vector, and allowing the expressedfusion protein to assemble much like antibody molecules, whereuponinterchain disulfide bonds form between the Fc moieties to yield thedimer.

The term “Fc polypeptide” as used herein includes native and muteinforms of polypeptides derived from the Fc region of an antibody.Truncated forms of such polypeptides containing the hinge region thatpromotes dimerization also are included. Fusion proteins comprising Fcmoieties (and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

One suitable Fc polypeptide, described in PCT application WO 93/10151and U.S. Pat. Nos. 5,426,048 and 5,262,522, is a single chainpolypeptide extending from the N-terminal hinge region to the nativeC-terminus of the Fc region of a human IgG1 antibody. Another useful Fcpolypeptide is the Fc mutein described in U.S. Pat. No. 5,457,035, andin Baum et al., (1994) EMBO J. 13:3992-4001. The amino acid sequence ofthis mutein is identical to that of the native Fc sequence presented inWO 93/10151, except that amino acid 19 has been changed from Leu to Ala,amino acid 20 has been changed from Leu to Glu, and amino acid 22 hasbeen changed from Gly to Ala. The mutein exhibits reduced affinity forFc receptors.

In other embodiments, the variable portion of the heavy and/or lightchains of a antigen binding protein such as disclosed herein can besubstituted for the variable portion of an antibody heavy and/or lightchain.

Alternatively, the oligomer is a fusion protein comprising multipleantigen binding proteins that specifically bind (i) β-Klotho; (ii)FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klothoand one of FGFR1c, FGFR2c, FGFR3c, and FGFR4, with or without peptidelinkers (spacer peptides). Among the suitable peptide linkers are thosedescribed in U.S. Pat. Nos. 4,751,180 and 4,935,233.

Another method for preparing oligomeric derivatives comprising thatantigen binding proteins that specifically bind (i) β-Klotho; (ii)FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klothoand one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 involves use of a leucinezipper. Leucine zipper domains are peptides that promote oligomerizationof the proteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., (1988)Science 240:1759), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble oligomericproteins are described in PCT application WO 94/10308, and the leucinezipper derived from lung surfactant protein D (SPD) described in Hoppeet al., (1994) FEBS Letters 344:191, hereby incorporated by reference.The use of a modified leucine zipper that allows for stabletrimerization of a heterologous protein fused thereto is described inFanslow et al., (1994) Semin. Immunol. 6:267-278. In one approach,recombinant fusion proteins comprising an antigen binding proteinfragment or derivative that specifically binds (i) β-Klotho; (ii)FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klothoand one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 is fused to a leucinezipper peptide are expressed in suitable host cells, and the solubleoligomeric antigen binding protein fragments or derivatives that formare recovered from the culture supernatant.

In certain embodiments, the antigen binding protein has a K_(D)(equilibrium binding affinity) of less than 1 pM, 10 pM, 100 pM, 1 nM, 2nM, 5 nM, 10 nM, 25 nM or 50 nM.

In another aspect the instant disclosure provides an antigen bindingprotein having a half-life of at least one day in vitro or in vivo(e.g., when administered to a human subject). In one embodiment, theantigen binding protein has a half-life of at least three days. Inanother embodiment, the antibody or portion thereof has a half-life offour days or longer. In another embodiment, the antibody or portionthereof has a half-life of eight days or longer. In another embodiment,the antibody or portion thereof has a half-life of ten days or longer.In another embodiment, the antibody or portion thereof has a half-lifeof eleven days or longer. In another embodiment, the antibody or portionthereof has a half-life of fifteen days or longer. In anotherembodiment, the antibody or antigen-binding portion thereof isderivatized or modified such that it has a longer half-life as comparedto the underivatized or unmodified antibody. In another embodiment, anantigen binding protein that specifically binds (i) β-Klotho; (ii)FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klothoand one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 contains point mutations toincrease serum half life, such as described in WO 00/09560, publishedFeb. 24, 2000, incorporated by reference.

Glycosylation

An antigen binding protein that specifically binds (i) β-Klotho; (ii)FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klothoand one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 can have a glycosylationpattern that is different or altered from that found in the nativespecies. As is known in the art, glycosylation patterns can depend onboth the sequence of the protein (e.g., the presence or absence ofparticular glycosylation amino acid residues, discussed below), or thehost cell or organism in which the protein is produced. Particularexpression systems are discussed below.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine can also be used.

Addition of glycosylation sites to the antigen binding protein isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tri-peptide sequences(for N-linked glycosylation sites). The alteration can also be made bythe addition of, or substitution by, one or more serine or threonineresidues to the starting sequence (for O-linked glycosylation sites).For ease, the antigen binding protein amino acid sequence can be alteredthrough changes at the DNA level, particularly by mutating the DNAencoding the target polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantigen binding protein is by chemical or enzymatic coupling ofglycosides to the protein. These procedures are advantageous in thatthey do not require production of the protein in a host cell that hasglycosylation capabilities for N- and O-linked glycosylation. Dependingon the coupling mode used, the sugar(s) can be attached to (a) arginineand histidine, (b) free carboxyl groups, (c) free sulfhydryl groups suchas those of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 and in Aplin andWriston, (1981) CRC Crit. Rev. Biochem., pp. 259-306.

Removal of carbohydrate moieties present on the starting antigen bindingprotein can be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the protein to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddinet al., (1987) Arch. Biochem. Biophys. 259:52 and by Edge et al., (1981)Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., (1987) Meth. Enzymol.138:350. Glycosylation at potential glycosylation sites can be preventedby the use of the compound tunicamycin as described by Duskin et al.,(1982) J. Biol. Chem. 257:3105. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Hence, aspects of the present disclosure include glycosylation variantsof antigen binding proteins that specifically bind (i) β-Klotho; (ii)FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klothoand one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 wherein the number and/ortype of glycosylation site(s) has been altered compared to the aminoacid sequences of the parent polypeptide. In certain embodiments,antibody protein variants comprise a greater or a lesser number ofN-linked glycosylation sites than the native antibody. An N-linkedglycosylation site is characterized by the sequence: Asn-X-Ser orAsn-X-Thr, wherein the amino acid residue designated as X can be anyamino acid residue except proline. The substitution of amino acidresidues to create this sequence provides a potential new site for theaddition of an N-linked carbohydrate chain. Alternatively, substitutionsthat eliminate or alter this sequence will prevent addition of anN-linked carbohydrate chain present in the native polypeptide. Forexample, the glycosylation can be reduced by the deletion of an Asn orby substituting the Asn with a different amino acid. In otherembodiments, one or more new N-linked sites are created. Antibodiestypically have a N-linked glycosylation site in the Fc region.

Labels and Effector Groups

In some embodiments, an antigen binding protein that specifically binds(i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4comprises one or more labels. The term “labeling group” or “label” meansany detectable label. Examples of suitable labeling groups include, butare not limited to the following: radioisotopes or radionuclides (e.g.,³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent groups(e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g.,horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase), chemiluminescent groups, biotinyl groups, or predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, epitope tags). In some embodiments, the labeling groupis coupled to the antigen binding protein via spacer arms of variouslengths to reduce potential steric hindrance. Various methods forlabeling proteins are known in the art and can be used as is seen fit.

The term “effector group” means any group coupled to an antigen bindingprotein that specifically binds one (i) β-Klotho; (ii) FGFR1c, FGFR2c,FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one ofFGFR1c, FGFR2c, FGFR3c, and FGFR4 that acts as a cytotoxic agent.Examples for suitable effector groups are radioisotopes or radionuclides(e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I). Other suitablegroups include toxins, therapeutic groups, or chemotherapeutic groups.Examples of suitable groups include calicheamicin, auristatins,geldanamycin and cantansine. In some embodiments, the effector group iscoupled to the antigen binding protein via spacer arms of variouslengths to reduce potential steric hindrance.

In general, labels fall into a variety of classes, depending on theassay in which they are to be detected: a) isotopic labels, which can beradioactive or heavy isotopes; b) magnetic labels (e.g., magneticparticles); c) redox active moieties; d) optical dyes; enzymatic groups(e.g. horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase); e) biotinylated groups; and f) predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags, etc.). In some embodiments, the labeling group iscoupled to the antigen binding protein via spacer arms of variouslengths to reduce potential steric hindrance. Various methods forlabeling proteins are known in the art.

Specific labels include optical dyes, including, but not limited to,chromophores, phosphors and fluorophores, with the latter being specificin many instances. Fluorophores can be either “small molecule” fluores,or proteinaceous fluores.

By “fluorescent label” is meant any molecule that can be detected viaits inherent fluorescent properties. Suitable fluorescent labelsinclude, but are not limited to, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue, TexasRed, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705,Oregon green, the Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430,Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594,Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680),Cascade Blue, Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes,Eugene, Oreg.), FITC, Rhodamine, and Texas Red (Pierce, Rockford, Ill.),Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.). Suitableoptical dyes, including fluorophores, are described in MOLECULAR PROBESHANDBOOK by Richard P. Haugland, hereby expressly incorporated byreference.

Suitable proteinaceous fluorescent labels also include, but are notlimited to, green fluorescent protein, including a Renilla, Ptilosarcus,or Aequorea species of GFP (Chalfie et al. (1994) Science 263:802-805),EGFP (Clontech Labs™, Inc., Genbank™ Accession Number U55762), bluefluorescent protein (BFP, Quantum Biotechnologies, Inc.™, Quebec,Canada; Stauber, (1998) Biotechniques 24:462-471; Heim et al., (1996)Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (EYFP™,Clontech Labs™, Inc.), luciferase™ (Ichiki et al., (1993) J.Immunol.150:5408-5417), β galactosidase (Nolan et al., (1988) Proc. Natl. Acad.Sci. U.S.A. 85:2603-2607) and Renilla (WO92/15673, WO95/07463,WO98/14605, WO98/26277, WO99/49019, U.S. Pat. Nos. 5,292,658, 5,418,155,5,683,888, 5,741,668, 5,777,079, 5,804,387, 5,874,304, 5,876,995,5,925,558).

Preparing Of Antigen Binding Proteins

Non-human antibodies that are provided can be, for example, derived fromany antibody-producing animal, such as mouse, rat, rabbit, goat, donkey,or non-human primate (such as monkey (e.g., cynomolgus or rhesus monkey)or ape (e.g., chimpanzee)). Non-human antibodies can be used, forinstance, in vitro cell culture and cell-culture based applications, orany other application where an immune response to the antibody does notoccur or is insignificant, can be prevented, is not a concern, or isdesired. In certain embodiments, the antibodies can be produced byimmunizing with full-length β-Klotho, FGFR1c, FGFR2c, FGFR3c or FGFR4(Example 1), with the extracellular domain of β-Klotho, FGFR1c, FGFR2c,FGFR3c or FGFR4 (Example 2), or two of β-Klotho, FGFR1c, FGFR2c, FGFR3cand FGFR4 (Example 1), with whole cells expressing FGFR1c, β-Klotho orboth FGFR1c and β-Klotho (Example 1 and 3), with membranes prepared fromcells expressing FGFR1c, β-Klotho or both FGFR1c and β-Klotho (Example 1and 3), with fusion proteins, e.g., Fc fusions comprising FGFR1c,β-Klotho or FGFR1c and β-Klotho (or extracellular domains thereof) fusedto Fc (Example 2 and 3), and other methods known in the art, e.g., asdescribed in the Examples presented herein. Alternatively, the certainnon-human antibodies can be raised by immunizing with amino acids whichare segments of one or more of β-Klotho, FGFR1c, FGFR2c, FGFR3c or FGFR4that form part of the epitope to which certain antibodies providedherein bind. The antibodies can be polyclonal, monoclonal, or can besynthesized in host cells by expressing recombinant DNA.

Fully human antibodies can be prepared as described above by immunizingtransgenic animals containing human immunoglobulin loci or by selectinga phage display library that is expressing a repertoire of humanantibodies.

The monoclonal antibodies (mAbs) can be produced by a variety oftechniques, including conventional monoclonal antibody methodology,e.g., the standard somatic cell hybridization technique of Kohler andMilstein, (1975) Nature 256:495. Alternatively, other techniques forproducing monoclonal antibodies can be employed, for example, the viralor oncogenic transformation of B-lymphocytes. One suitable animal systemfor preparing hybridomas is the murine system, which is a very wellestablished procedure. Immunization protocols and techniques forisolation of immunized splenocytes for fusion are known in the art. Forsuch procedures, B cells from immunized mice are fused with a suitableimmortalized fusion partner, such as a murine myeloma cell line. Ifdesired, rats or other mammals besides can be immunized instead of miceand B cells from such animals can be fused with the murine myeloma cellline to form hybridomas. Alternatively, a myeloma cell line from asource other than mouse can be used. Fusion procedures for makinghybridomas also are well known. SLAM technology can also be employed inthe production of antibodies.

The single chain antibodies that are provided can be formed by linkingheavy and light chain variable domain (Fv region) fragments via an aminoacid bridge (short peptide linker), resulting in a single polypeptidechain. Such single-chain Fvs (scFvs) can be prepared by fusing DNAencoding a peptide linker between DNAs encoding the two variable domainpolypeptides (V_(L) and V_(H)). The resulting polypeptides can fold backon themselves to form antigen-binding monomers, or they can formmultimers (e.g., dimers, trimers, or tetramers), depending on the lengthof a flexible linker between the two variable domains (Kortt et al.,(1997) Prot. Eng. 10:423; Kortt et al., (2001) Biomol. Eng. 18:95-108).By combining different V_(L) and V_(H)-comprising polypeptides, one canform multimeric scFvs that bind to different epitopes (Kriangkum et al.,(2001) Biomol. Eng. 18:31-40). Techniques developed for the productionof single chain antibodies include those described in U.S. Pat. No.4,946,778; Bird, (1988) Science 242:423; Huston et al., (1988) Proc.Natl. Acad. Sci. U.S.A. 85:5879; Ward et al., (1989) Nature 334:544, deGraaf et al., (2002) Methods Mol. Biol. 178:379-387. Single chainantibodies derived from antibodies provided herein include, but are notlimited to scFvs comprising the variable domain combinations of theheavy and light chain variable regions depicted in Table 2, orcombinations of light and heavy chain variable domains which includeCDRs depicted in Tables 3 and 4.

Antibodies provided herein that are of one subclass can be changed toantibodies from a different subclass using subclass switching methods.Thus, IgG antibodies can be derived from an IgM antibody, for example,and vice versa. Such techniques allow the preparation of new antibodiesthat possess the antigen binding properties of a given antibody (theparent antibody), but also exhibit biological properties associated withan antibody isotype or subclass different from that of the parentantibody. Recombinant DNA techniques can be employed. Cloned DNAencoding particular antibody polypeptides can be employed in suchprocedures, e.g., DNA encoding the constant domain of an antibody of thedesired isotype. See, e.g., Lantto et al., (2002) Methods Mol. Biol.178:303-316.

Accordingly, the antibodies that are provided include those comprising,for example, the variable domain combinations described, supra., havinga desired isotype (for example, IgA, IgG1, IgG2, IgG3, IgG4, IgE, andIgD) as well as Fab or F(ab′)₂ fragments thereof. Moreover, if an IgG4is desired, it can also be desired to introduce a point mutation(CPSCP->CPPCP (SEQ ID NOS 380-381, respectively, in order ofappearance)) in the hinge region as described in Bloom et al., (1997)Protein Science 6:407, incorporated by reference herein) to alleviate atendency to form intra-H chain disulfide bonds that can lead toheterogeneity in the IgG4 antibodies.

Moreover, techniques for deriving antibodies having different properties(i.e., varying affinities for the antigen to which they bind) are alsoknown. One such technique, referred to as chain shuffling, involvesdisplaying immunoglobulin variable domain gene repertoires on thesurface of filamentous bacteriophage, often referred to as phagedisplay. Chain shuffling has been used to prepare high affinityantibodies to the hapten 2-phenyloxazol-5-one, as described by Marks etal., (1992) BioTechnology 10:779.

Conservative modifications can be made to the heavy and light chainvariable regions described in Table 2, or the CDRs described in Tables3A and 3B, 4A and 4B, and Table 6C, infra (and correspondingmodifications to the encoding nucleic acids) to produce an antigenbinding protein having functional and biochemical characteristics.Methods for achieving such modifications are described above.

Antigen binding proteins that specifically bind one or more of (i)β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 can befurther modified in various ways. For example, if they are to be usedfor therapeutic purposes, they can be conjugated with polyethyleneglycol (PEGylated) to prolong the serum half-life or to enhance proteindelivery. Alternatively, the V region of the subject antibodies orfragments thereof can be fused with the Fc region of a differentantibody molecule. The Fc region used for this purpose can be modifiedso that it does not bind complement, thus reducing the likelihood ofinducing cell lysis in the patient when the fusion protein is used as atherapeutic agent. In addition, the subject antibodies or functionalfragments thereof can be conjugated with human serum albumin to enhancethe serum half-life of the antibody or fragment thereof. Another usefulfusion partner for the antigen binding proteins or fragments thereof istransthyretin (TTR). TTR has the capacity to form a tetramer, thus anantibody-TTR fusion protein can form a multivalent antibody which canincrease its binding avidity.

Alternatively, substantial modifications in the functional and/orbiochemical characteristics of the antigen binding proteins describedherein can be achieved by creating substitutions in the amino acidsequence of the heavy and light chains that differ significantly intheir effect on maintaining (a) the structure of the molecular backbonein the area of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulkiness of the side chain. A “conservativeamino acid substitution” can involve a substitution of a native aminoacid residue with a normative residue that has little or no effect onthe polarity or charge of the amino acid residue at that position. See,Table 5, supra. Furthermore, any native residue in the polypeptide canalso be substituted with alanine, as has been previously described foralanine scanning mutagenesis.

Amino acid substitutions (whether conservative or non-conservative) ofthe subject antibodies can be implemented by those skilled in the art byapplying routine techniques. Amino acid substitutions can be used toidentify important residues of the antibodies provided herein, or toincrease or decrease the affinity of these antibodies for one or more of(i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 or formodifying the binding affinity of other antigen-binding proteinsdescribed herein.

Methods of Expressing Antigen Binding Proteins

Expression systems and constructs in the form of plasmids, expressionvectors, transcription or expression cassettes that comprise at leastone polynucleotide as described above are also provided herein, as wellhost cells comprising such expression systems or constructs.

The antigen binding proteins provided herein can be prepared by any of anumber of conventional techniques. For example, antigen binding proteinsthat specifically bind (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c orFGFR4; or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c,FGFR3c, and FGFR4 can be produced by recombinant expression systems,using any technique known in the art. See, e.g., Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.)Plenum Press, New York (1980); and Antibodies: A Laboratory Manual,Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1988).

Antigen binding proteins can be expressed in hybridoma cell lines (e.g.,in particular antibodies can be expressed in hybridomas) or in celllines other than hybridomas. Expression constructs encoding theantibodies can be used to transform a mammalian, insect or microbialhost cell. Transformation can be performed using any known method forintroducing polynucleotides into a host cell, including, for examplepackaging the polynucleotide in a virus or bacteriophage and transducinga host cell with the construct by transfection procedures known in theart, as exemplified by U.S. Pat. Nos. 4,399,216; 4,912,040; 4,740,461;4,959,455. The optimal transformation procedure used will depend uponwhich type of host cell is being transformed. Methods for introductionof heterologous polynucleotides into mammalian cells are well known inthe art and include, but are not limited to, dextran-mediatedtransfection, calcium phosphate precipitation, polybrene mediatedtransfection, protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, mixing nucleic acid withpositively-charged lipids, and direct microinjection of the DNA intonuclei.

Recombinant expression constructs typically comprise a nucleic acidmolecule encoding a polypeptide comprising one or more of the following:one or more CDRs provided herein; a light chain constant region; a lightchain variable region; a heavy chain constant region (e.g., C_(H)1,C_(H)2 and/or C_(H)3); and/or another scaffold portion of an antigenbinding protein. These nucleic acid sequences are inserted into anappropriate expression vector using standard ligation techniques. In oneembodiment, the heavy or light chain constant region is appended to theC-terminus of the anti-β-Klotho, -FGFR1c, -FGFR2c, -FGFR3c, -FGFR4, orβ-Klotho and FGFR1c-specific heavy or light chain variable region and isligated into an expression vector. The vector is typically selected tobe functional in the particular host cell employed (i.e., the vector iscompatible with the host cell machinery, permitting amplification and/orexpression of the gene can occur). In some embodiments, vectors are usedthat employ protein-fragment complementation assays using proteinreporters, such as dihydrofolate reductase (see, for example, U.S. Pat.No. 6,270,964, which is hereby incorporated by reference). Suitableexpression vectors can be purchased, for example, from Invitrogen LifeTechnologies or BD Biosciences (formerly “Clontech”). Other usefulvectors for cloning and expressing the antibodies and fragments includethose described in Bianchi and McGrew, (2003) Biotech. Biotechnol.Bioeng. 84:439-44, which is hereby incorporated by reference. Additionalsuitable expression vectors are discussed, for example, in MethodsEnzymol., vol. 185 (D. V. Goeddel, ed.), 1990, New York: Academic Press.

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” in certain embodiments will typically includeone or more of the following nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these sequences is discussed below.

Optionally, the vector can contain a “tag”-encoding sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of an antigenbinding protein coding sequence; the oligonucleotide sequence encodespolyHis (such as hexaHis (SEQ ID NO: 382)), or another “tag” such asFLAG, HA (hemaglutinin influenza virus), or myc, for which commerciallyavailable antibodies exist. This tag is typically fused to thepolypeptide upon expression of the polypeptide, and can serve as a meansfor affinity purification or detection of the antigen binding proteinfrom the host cell. Affinity purification can be accomplished, forexample, by column chromatography using antibodies against the tag as anaffinity matrix. Optionally, the tag can subsequently be removed fromthe purified antigen binding protein by various means such as usingcertain peptidases for cleavage.

Flanking sequences can be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), synthetic or native. Assuch, the source of a flanking sequence can be any prokaryotic oreukaryotic organism, any vertebrate or invertebrate organism, or anyplant, provided that the flanking sequence is functional in, and can beactivated by, the host cell machinery.

Flanking sequences useful in the vectors can be obtained by any ofseveral methods well known in the art. Typically, flanking sequencesuseful herein will have been previously identified by mapping and/or byrestriction endonuclease digestion and can thus be isolated from theproper tissue source using the appropriate restriction endonucleases. Insome cases, the full nucleotide sequence of a flanking sequence can beknown. Here, the flanking sequence can be synthesized using the methodsdescribed herein for nucleic acid synthesis or cloning.

Whether all or only a portion of the flanking sequence is known, it canbe obtained using polymerase chain reaction (PCR) and/or by screening agenomic library with a suitable probe such as an oligonucleotide and/orflanking sequence fragment from the same or another species. Where theflanking sequence is not known, a fragment of DNA containing a flankingsequence can be isolated from a larger piece of DNA that can contain,for example, a coding sequence or even another gene or genes. Isolationcan be accomplished by restriction endonuclease digestion to produce theproper DNA fragment followed by isolation using agarose gelpurification, Qiagen® column chromatography (Chatsworth, Calif.), orother methods known to the skilled artisan. The selection of suitableenzymes to accomplish this purpose will be readily apparent to one ofordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. If the vector of choice doesnot contain an origin of replication site, one can be chemicallysynthesized based on a known sequence, and ligated into the vector. Forexample, the origin of replication from the plasmid pBR322 (New EnglandBiolabs, Beverly, Mass.) is suitable for most gram-negative bacteria,and various viral origins (e.g., SV40, polyoma, adenovirus, vesicularstomatitus virus (VSV), or papillomaviruses such as HPV or BPV) areuseful for cloning vectors in mammalian cells. Generally, the origin ofreplication component is not needed for mammalian expression vectors(for example, the SV40 origin is often used only because it alsocontains the virus early promoter).

A transcription termination sequence is typically located 3′ to the endof a polypeptide coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly-T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene encodes a protein necessary for the survivaland growth of a host cell grown in a selective culture medium. Typicalselection marker genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, tetracycline, orkanamycin for prokaryotic host cells; (b) complement auxotrophicdeficiencies of the cell; or (c) supply critical nutrients not availablefrom complex or defined media. Specific selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. Advantageously, a neomycin resistance genecan also be used for selection in both prokaryotic and eukaryotic hostcells.

Other selectable genes can be used to amplify the gene that will beexpressed. Amplification is the process wherein genes that are requiredfor production of a protein critical for growth or cell survival arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and promoterless thymidinekinase genes. Mammalian cell transformants are placed under selectionpressure wherein only the transformants are uniquely adapted to surviveby virtue of the selectable gene present in the vector. Selectionpressure is imposed by culturing the transformed cells under conditionsin which the concentration of selection agent in the medium issuccessively increased, thereby leading to the amplification of both theselectable gene and the DNA that encodes another gene, such as anantigen binding protein that binds (i) β-Klotho; (ii) FGFR1c, FGFR2c,FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one ofFGFR1c, FGFR2c, FGFR3c, and FGFR4. As a result, increased quantities ofa polypeptide such as an antigen binding protein are synthesized fromthe amplified DNA.

A ribosome-binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the polypeptide to beexpressed.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one can manipulate the various pre- orpro-sequences to improve glycosylation or yield. For example, one canalter the peptidase cleavage site of a particular signal peptide, or addprosequences, which also can affect glycosylation. The final proteinproduct can have, in the −1 position (relative to the first amino acidof the mature protein), one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product can have one or two amino acid residues found inthe peptidase cleavage site, attached to the amino-terminus.Alternatively, use of some enzyme cleavage sites can result in aslightly truncated form of the desired polypeptide, if the enzyme cutsat such area within the mature polypeptide.

Expression and cloning will typically contain a promoter that isrecognized by the host organism and operably linked to the moleculeencoding an antigen binding protein that specifically binds (i)β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4.Promoters are untranscribed sequences located upstream (i.e., 5′) to thestart codon of a structural gene (generally within about 100 to 1000 bp)that control transcription of the structural gene. Promoters areconventionally grouped into one of two classes: inducible promoters andconstitutive promoters. Inducible promoters initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, such as the presence or absence of a nutrient or achange in temperature. Constitutive promoters, on the other hand,uniformly transcribe a gene to which they are operably linked, that is,with little or no control over gene expression. A large number ofpromoters, recognized by a variety of potential host cells, are wellknown. A suitable promoter is operably linked to the DNA encoding heavychain or light chain comprising an antigen binding protein by removingthe promoter from the source DNA by restriction enzyme digestion andinserting the desired promoter sequence into the vector.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retroviruses, hepatitis-B virus, and Simian Virus 40(SV40). Other suitable mammalian promoters include heterologousmammalian promoters, for example, heat-shock promoters and the actinpromoter.

Additional promoters which can be of interest include, but are notlimited to: SV40 early promoter (Benoist and Chambon, (1981) Nature290:304-310); CMV promoter (Thornsen et al., (1984) Proc. Natl. Acad.U.S.A. 81:659-663); the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto et al., (1980) Cell 22:787-797);herpes thymidine kinase promoter (Wagner et al., (1981) Proc. Natl.Acad. Sci. U.S.A. 78:1444-1445); promoter and regulatory sequences fromthe metallothionine gene (Prinster et al., (1982) Nature 296:39-42); andprokaryotic promoters such as the beta-lactamase promoter(VIIIa-Kamaroff et al., (1978) Proc. Natl. Acad. Sci. U.S.A.75:3727-3731); or the tac promoter (DeBoer et al., (1983) Proc. Natl.Acad. Sci. U.S.A. 80:21-25). Also of interest are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion that is active in pancreatic acinar cells (Swift et al., (1984)Cell 38:639-646; Ornitz et al., (1986) Cold Spring Harbor Symp. Quant.Biol. 50:399-409; MacDonald, (1987) Hepatology 7:425-515); the insulingene control region that is active in pancreatic beta cells (Hanahan,(1985) Nature 315:115-122); the immunoglobulin gene control region thatis active in lymphoid cells (Grosschedl et al., (1984) Cell 38:647-658;Adames et al., (1985) Nature 318:533-538; Alexander et al., (1987) Mol.Cell. Biol. 7:1436-1444); the mouse mammary tumor virus control regionthat is active in testicular, breast, lymphoid and mast cells (Leder etal., (1986) Cell 45:485-495); the albumin gene control region that isactive in liver (Pinkert et al., (1987) Genes and Devel. 1:268-276); thealpha-feto-protein gene control region that is active in liver (Krumlaufet al., (1985) Mol. Cell. Biol. 5:1639-1648; Hammer et al., (1987)Science 253:53-58); the alpha 1-antitrypsin gene control region that isactive in liver (Kelsey et al., (1987) Genes and Devel. 1:161-171); thebeta-globin gene control region that is active in myeloid cells (Mogramet al., (1985) Nature 315:338-340; Kollias et al., (1986) Cell46:89-94); the myelin basic protein gene control region that is activein oligodendrocyte cells in the brain (Readhead et al., (1987) Cell48:703-712); the myosin light chain-2 gene control region that is activein skeletal muscle (Sani, (1985) Nature 314:283-286); and thegonadotropic releasing hormone gene control region that is active in thehypothalamus (Mason et al., (1986) Science 234:1372-1378).

An enhancer sequence can be inserted into the vector to increasetranscription of DNA encoding light chain or heavy chain comprising anantigen binding protein that specifically binds (i) β-Klotho; (ii)FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klothoand one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 by higher eukaryotes, e.g.,a human antigen binding protein that specifically binds (i) β-Klotho;(ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprisingβ-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4. Enhancers arecis-acting elements of DNA, usually about 10-300 by in length, that acton the promoter to increase transcription. Enhancers are relativelyorientation and position independent, having been found at positionsboth 5′ and 3′ to the transcription unit. Several enhancer sequencesavailable from mammalian genes are known (e.g., globin, elastase,albumin, alpha-feto-protein and insulin). Typically, however, anenhancer from a virus is used. The SV40 enhancer, the cytomegalovirusearly promoter enhancer, the polyoma enhancer, and adenovirus enhancersknown in the art are exemplary enhancing elements for the activation ofeukaryotic promoters. While an enhancer can be positioned in the vectoreither 5′ or 3′ to a coding sequence, it is typically located at a site5′ from the promoter. A sequence encoding an appropriate native orheterologous signal sequence (leader sequence or signal peptide) can beincorporated into an expression vector, to promote extracellularsecretion of the antibody. The choice of signal peptide or leaderdepends on the type of host cells in which the antibody is to beproduced, and a heterologous signal sequence can replace the nativesignal sequence. Examples of signal peptides that are functional inmammalian host cells include the following: the signal sequence forinterleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195; the signalsequence for interleukin-2 receptor described in Cosman et al., (1984)Nature 312:768; the interleukin-4 receptor signal peptide described inEP Patent No. 0367 566; the type I interleukin-1 receptor signal peptidedescribed in U.S. Pat. No. 4,968,607; the type II interleukin-1 receptorsignal peptide described in EP Patent No. 0 460 846.

The expression vectors that are provided can be constructed from astarting vector such as a commercially available vector. Such vectorscan but need not contain all of the desired flanking sequences. Whereone or more of the flanking sequences described herein are not alreadypresent in the vector, they can be individually obtained and ligatedinto the vector. Methods used for obtaining each of the flankingsequences are well known to one skilled in the art.

After the vector has been constructed and a nucleic acid moleculeencoding light chain, a heavy chain, or a light chain and a heavy chaincomprising an antigen binding protein that specifically binds (i)β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 hasbeen inserted into the proper site of the vector, the completed vectorcan be inserted into a suitable host cell for amplification and/orpolypeptide expression. The transformation of an expression vector foran antigen binding protein into a selected host cell can be accomplishedby well known methods including transfection, infection, calciumphosphate co-precipitation, electroporation, microinjection,lipofection, DEAE-dextran mediated transfection, or other knowntechniques. The method selected will in part be a function of the typeof host cell to be used. These methods and other suitable methods arewell known to the skilled artisan, and are set forth, for example, inSambrook et al., (2001), supra.

A host cell, when cultured under appropriate conditions, synthesizes anantigen binding protein that can subsequently be collected from theculture medium (if the host cell secretes it into the medium) ordirectly from the host cell producing it (if it is not secreted). Theselection of an appropriate host cell will depend upon various factors,such as desired expression levels, polypeptide modifications that aredesirable or necessary for activity (such as glycosylation orphosphorylation) and ease of folding into a biologically activemolecule.

Mammalian cell lines available as hosts for expression are well known inthe art and include, but are not limited to, immortalized cell linesavailable from the American Type Culture Collection (ATCC), includingbut not limited to Chinese hamster ovary (CHO) cells, HeLa cells, babyhamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), and a number of othercell lines. In certain embodiments, cell lines can be selected throughdetermining which cell lines have high expression levels andconstitutively produce antigen binding proteins with desirable bindingproperties (e.g., the ability to bind (i) β-Klotho; (ii) FGFR1c, FGFR2c,FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one ofFGFR1c, FGFR2c, FGFR3c, and FGFR4). In another embodiment, a cell linefrom the B cell lineage that does not make its own antibody but has acapacity to make and secrete a heterologous antibody can be selected.The ability to induce FGF21-like signaling can also form a selectioncriterion.

Uses of Antigen Binding Proteins for Diagnostic and Therapeutic Purposes

The antigen binding proteins disclosed herein are useful for detecting(i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 inbiological samples and identification of cells or tissues that produceone or more of (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or(iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c,and FGFR4. For instance, the antigen binding proteins disclosed hereincan be used in diagnostic assays, e.g., binding assays to detect and/orquantify (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) acomplex comprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4expressed in a tissue or cell. Antigen binding proteins thatspecifically bind to (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4;or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c,FGFR3c, and FGFR4 can be used in treatment of diseases related toFGF21-like signaling in a patient in need thereof, such as type 2diabetes, obesity, dyslipidemia, NASH, cardiovascular disease, andmetabolic syndrome. By forming a signaling complex comprising an antigenbinding protein, and (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4;or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c,FGFR3c, and FGFR4, the natural in vivo activity of FGF21, whichassociates with FGFR1c, FGFR2c, FGFR3c, FGFR4 and β-Klotho in vivo toinitiate signaling, can be mimicked and/or enchanced, leading totherapeutic effects.

Indications

A disease or condition associated with human FGF21 includes any diseaseor condition whose onset in a patient is caused by, at least in part,the induction of FGF21-like signaling, which is initiated in vivo by theformation of a complex comprising FGFR1c, FGFR2c, FGFR3c or FGFR4 andβ-Klotho and FGF21. The severity of the disease or condition can also bedecreased by the induction of FGF21-like signaling. Examples of diseasesand conditions that can be treated with the antigen binding proteinsinclude type 2 diabetes, obesity, dyslipidemia, NASH, cardiovasculardisease, and metabolic syndrome.

The antigen binding proteins described herein can be used to treat type2 diabetes, obesity, dyslipidemia, NASH, cardiovascular disease, andmetabolic syndrome, or can be employed as a prophylactic treatmentadministered, e.g., daily, weekly, biweekly, monthly, bimonthly,biannually, etc to prevent or reduce the frequency and/or severity ofsymptoms, e.g., elevated plasma glucose levels, elevated triglyceridesand cholesterol levels, thereby providing an improved glycemic andcardiovascular risk factor profile.

Diagnostic Methods

The antigen binding proteins described herein can be used for diagnosticpurposes to detect, diagnose, or monitor diseases and/or conditionsassociated with FGFR1c, FGFR2c, FGFR3c, FGFR4, β-Klotho, FGF21 orcombinations thereof. Also provided are methods for the detection of thepresence of (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii)a complex comprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, andFGFR4 in a sample using classical immunohistological methods known tothose of skill in the art (e.g., Tijssen, 1993, Practice and Theory ofEnzyme Immunoassays, Vol 15 (Eds R. H. Burdon and P. H. van Knippenberg,Elsevier, Amsterdam); Zola, (1987) Monoclonal Antibodies: A Manual ofTechniques, pp. 147-158 (CRC Press, Inc.); Jalkanen et al., (1985) J.Cell. Biol. 101:976-985; Jalkanen et al., (1987) J. Cell Biol.105:3087-3096). The detection of (i) β-Klotho; (ii) FGFR1c, FGFR2c,FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one ofFGFR1c, FGFR2c, FGFR3c, and FGFR4 can be performed in vivo or in vitro.

Diagnostic applications provided herein include use of the antigenbinding proteins to detect expression of (i) β-Klotho; (ii) FGFR1c,FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and oneof FGFR1c, FGFR2c, FGFR3c, and FGFR4 and/or binding to (i) β-Klotho;(ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprisingβ-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4. Examples ofmethods useful in the detection of the presence of (i) β-Klotho; (ii)FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klothoand one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 include immunoassays, suchas the enzyme linked immunosorbent assay (ELISA) and theradioimmunoassay (RIA).

For diagnostic applications, the antigen binding protein typically willbe labeled with a detectable labeling group. Suitable labeling groupsinclude, but are not limited to, the following: radioisotopes orradionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I),fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors),enzymatic groups (e.g., horseradish peroxidase, β-galactosidase,luciferase, alkaline phosphatase), chemiluminescent groups, biotinylgroups, or predetermined polypeptide epitopes recognized by a secondaryreporter (e.g., leucine zipper pair sequences, binding sites forsecondary antibodies, metal binding domains, epitope tags). In someembodiments, the labeling group is coupled to the antigen bindingprotein via spacer arms of various lengths to reduce potential sterichindrance. Various methods for labeling proteins are known in the artand can be used.

In another aspect, an antigen binding protein can be used to identify acell or cells that express (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c orFGFR4; or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c,FGFR3c, and FGFR4. In a specific embodiment, the antigen binding proteinis labeled with a labeling group and the binding of the labeled antigenbinding protein to (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4;or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c,FGFR3c, and FGFR4 is detected. In a further specific embodiment, thebinding of the antigen binding protein to (i) β-Klotho; (ii) FGFR1c,FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and oneof FGFR1c, FGFR2c, FGFR3c, and FGFR4 detected in vivo. In a furtherspecific embodiment, the antigen binding protein is isolated andmeasured using techniques known in the art. See, for example, Harlow andLane, (1988) Antibodies: A Laboratory Manual, New York: Cold SpringHarbor (ed. 1991 and periodic supplements); John E. Coligan, ed., (1993)Current Protocols In Immunology New York: John Wiley & Sons.

Another aspect provides for detecting the presence of a test moleculethat competes for binding to (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3cor FGFR4; or (iii) a complex comprising β-Klotho and one of FGFR1c,FGFR2c, FGFR3c, and FGFR4 with the antigen binding proteins provided, asdisclosed herein. An example of one such assay could involve detectingthe amount of free antigen binding protein in a solution containing anamount of one or more of (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c orFGFR4; or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c,FGFR3c, and FGFR4 in the presence or absence of the test molecule. Anincrease in the amount of free antigen binding protein (i.e., theantigen binding protein not bound to (i) β-Klotho; (ii) FGFR1c, FGFR2c,FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one ofFGFR1c, FGFR2c, FGFR3c, and FGFR4) would indicate that the test moleculeis capable of competing for binding to (i) β-Klotho; (ii) FGFR1c,FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and oneof FGFR1c, FGFR2c, FGFR3c, and FGFR4 with the antigen binding protein.In one embodiment, the antigen binding protein is labeled with alabeling group. Alternatively, the test molecule is labeled and theamount of free test molecule is monitored in the presence and absence ofan antigen binding protein.

Methods of Treatment: Pharmaceutical Formulations and Routes ofAdministration

Methods of using the antigen binding proteins are also provided. In somemethods, an antigen binding protein is provided to a patient. Theantigen binding protein induces FGF21-like signaling.

Pharmaceutical compositions that comprise a therapeutically effectiveamount of one or a plurality of the antigen binding proteins and apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative, and/or adjuvant are also provided. In addition, methods oftreating a patient by administering such pharmaceutical composition areincluded. The term “patient” includes human patients.

Acceptable formulation materials are nontoxic to recipients at thedosages and concentrations employed. In specific embodiments,pharmaceutical compositions comprising a therapeutically effectiveamount of human antigen binding proteins that specifically bind (i)β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 areprovided.

In certain embodiments, acceptable formulation materials preferably arenontoxic to recipients at the dosages and concentrations employed. Incertain embodiments, the pharmaceutical composition can containformulation materials for modifying, maintaining or preserving, forexample, the pH, osmolarity, viscosity, clarity, color, isotonicity,odor, sterility, stability, rate of dissolution or release, adsorptionor penetration of the composition. In such embodiments, suitableformulation materials include, but are not limited to, amino acids (suchas glycine, glutamine, asparagine, arginine or lysine); antimicrobials;antioxidants (such as ascorbic acid, sodium sulfite or sodiumhydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl,citrates, phosphates or other organic acids); bulking agents (such asmannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as Pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants. See,Remington's Pharmaceutical Sciences, 18th Edition, (A. R. Gennaro, ed.),1990, Mack Publishing Company.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, Remington's Pharmaceutical Sciences, supra. In certainembodiments, such compositions can influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theantigen binding proteins disclosed. In certain embodiments, the primaryvehicle or carrier in a pharmaceutical composition can be either aqueousor non-aqueous in nature. For example, a suitable vehicle or carrier canbe water for injection, physiological saline solution or artificialcerebrospinal fluid, possibly supplemented with other materials commonin compositions for parenteral administration. Neutral buffered salineor saline mixed with serum albumin are further exemplary vehicles. Inspecific embodiments, pharmaceutical compositions comprise Tris bufferof about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, and canfurther include sorbitol or a suitable substitute. In certainembodiments, compositions comprising antigen binding proteins thatspecifically bind (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or(iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c,and FGFR4 can be prepared for storage by mixing the selected compositionhaving the desired degree of purity with optional formulation agents(Remington's Pharmaceutical Sciences, supra) in the form of alyophilized cake or an aqueous solution. Further, in certainembodiments, antigen binding protein that bind (i) β-Klotho; (ii)FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klothoand one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 can be formulated as alyophilizate using appropriate excipients such as sucrose.

The pharmaceutical compositions can be selected for parenteral delivery.Alternatively, the compositions can be selected for inhalation or fordelivery through the digestive tract, such as orally. Preparation ofsuch pharmaceutically acceptable compositions is within the skill of theart.

The formulation components are present preferably in concentrations thatare acceptable to the site of administration. In certain embodiments,buffers are used to maintain the composition at physiological pH or at aslightly lower pH, typically within a pH range of from about 5 to about8.

When parenteral administration is contemplated, the therapeuticcompositions can be provided in the form of a pyrogen-free, parenterallyacceptable aqueous solution comprising the desired antigen bindingprotein in a pharmaceutically acceptable vehicle. A particularlysuitable vehicle for parenteral injection is sterile distilled water inwhich the antigen binding protein is formulated as a sterile, isotonicsolution, properly preserved. In certain embodiments, the preparationcan involve the formulation of the desired molecule with an agent, suchas injectable microspheres, bio-erodible particles, polymeric compounds(such as polylactic acid or polyglycolic acid), beads or liposomes, thatcan provide controlled or sustained release of the product which can bedelivered via depot injection. In certain embodiments, hyaluronic acidcan also be used, which can have the effect of promoting sustainedduration in the circulation. In certain embodiments, implantable drugdelivery devices can be used to introduce the desired antigen bindingprotein.

Certain pharmaceutical compositions are formulated for inhalation. Insome embodiments, antigen binding proteins that bind to (i) β-Klotho;(ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprisingβ-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 are formulated asa dry, inhalable powder. In specific embodiments, antigen bindingprotein inhalation solutions can also be formulated with a propellantfor aerosol delivery. In certain embodiments, solutions can benebulized. Pulmonary administration and formulation methods thereforeare further described in International Patent Application No.PCT/US94/001875, which is incorporated by reference and describespulmonary delivery of chemically modified proteins. Some formulationscan be administered orally. Antigen binding proteins that specificallybind (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) acomplex comprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4that are administered in this fashion can be formulated with or withoutcarriers customarily used in the compounding of solid dosage forms suchas tablets and capsules. In certain embodiments, a capsule can bedesigned to release the active portion of the formulation at the pointin the gastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional agents can be includedto facilitate absorption of an antigen binding protein. Diluents,flavorings, low melting point waxes, vegetable oils, lubricants,suspending agents, tablet disintegrating agents, and binders can also beemployed.

Some pharmaceutical compositions comprise an effective quantity of oneor a plurality of human antigen binding proteins that specifically bind(i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 in amixture with non-toxic excipients that are suitable for the manufactureof tablets. By dissolving the tablets in sterile water, or anotherappropriate vehicle, solutions can be prepared in unit-dose form.Suitable excipients include, but are not limited to, inert diluents,such as calcium carbonate, sodium carbonate or bicarbonate, lactose, orcalcium phosphate; or binding agents, such as starch, gelatin, oracacia; or lubricating agents such as magnesium stearate, stearic acid,or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving antigen binding proteinsthat specifically bind (i) β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c orFGFR4; or (iii) a complex comprising β-Klotho and one of FGFR1c, FGFR2c,FGFR3c, and FGFR4 in sustained- or controlled-delivery formulations.Techniques for formulating a variety of other sustained- orcontrolled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art. See, for example, International PatentApplication No. PCT/US93/00829, which is incorporated by reference anddescribes controlled release of porous polymeric microparticles fordelivery of pharmaceutical compositions. Sustained-release preparationscan include semipermeable polymer matrices in the form of shapedarticles, e.g., films, or microcapsules. Sustained release matrices caninclude polyesters, hydrogels, polylactides (as disclosed in U.S. Pat.No. 3,773,919 and European Patent Application Publication No. EP 058481,each of which is incorporated by reference), copolymers of L-glutamicacid and gamma ethyl-L-glutamate (Sidman et al., 1983, Biopolymers2:547-556), poly (2-hydroxyethyl-inethacrylate) (Langer et al., 1981, J.Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105),ethylene vinyl acetate (Langer et al., 1981, supra) orpoly-D(−)-3-hydroxybutyric acid (European Patent Application PublicationNo. EP 133,988). Sustained release compositions can also includeliposomes that can be prepared by any of several methods known in theart. See, e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. U.S.A.82:3688-3692; European Patent Application Publication Nos. EP 036,676;EP 088,046 and EP 143,949, incorporated by reference.

Pharmaceutical compositions used for in vivo administration aretypically provided as sterile preparations. Sterilization can beaccomplished by filtration through sterile filtration membranes. Whenthe composition is lyophilized, sterilization using this method can beconducted either prior to or following lyophilization andreconstitution. Compositions for parenteral administration can be storedin lyophilized form or in a solution. Parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

In certain embodiments, cells expressing a recombinant antigen bindingprotein as disclosed herein is encapsulated for delivery (see, Invest.Ophthalmol Vis Sci (2002) 43:3292-3298 and Proc. Natl. Acad. SciencesUSA (2006) 103:3896-3901).

In certain formulations, an antigen binding protein has a concentrationof at least 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml,70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml or 150 mg/ml. Some formulationscontain a buffer, sucrose and polysorbate. An example of a formulationis one containing 50-100 mg/ml of antigen binding protein, 5-20 mMsodium acetate, 5-10% w/v sucrose, and 0.002-0.008% w/v polysorbate.Certain, formulations, for instance, contain 65-75 mg/ml of an antigenbinding protein in 9-11 mM sodium acetate buffer, 8-10% w/v sucrose, and0.005-0.006% w/v polysorbate. The pH of certain such formulations is inthe range of 4.5-6. Other formulations have a pH of 5.0-5.5 (e.g., pH of5.0, 5.2 or 5.4).

Once the pharmaceutical composition has been formulated, it can bestored in sterile vials as a solution, suspension, gel, emulsion, solid,crystal, or as a dehydrated or lyophilized powder. Such formulations canbe stored either in a ready-to-use form or in a form (e.g., lyophilized)that is reconstituted prior to administration. Kits for producing asingle-dose administration unit are also provided. Certain kits containa first container having a dried protein and a second container havingan aqueous formulation. In certain embodiments, kits containing singleand multi-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are provided. The therapeutically effective amount of anantigen binding protein-containing pharmaceutical composition to beemployed will depend, for example, upon the therapeutic context andobjectives. One skilled in the art will appreciate that the appropriatedosage levels for treatment will vary depending, in part, upon themolecule delivered, the indication for which the antigen binding proteinis being used, the route of administration, and the size (body weight,body surface or organ size) and/or condition (the age and generalhealth) of the patient. In certain embodiments, the clinician can titerthe dosage and modify the route of administration to obtain the optimaltherapeutic effect.

A typical dosage can range from about 1 μg/kg to up to about 30 mg/kg ormore, depending on the factors mentioned above. In specific embodiments,the dosage can range from 10 μg/kg up to about 30 mg/kg, optionally from0.1 mg/kg up to about 30 mg/kg, alternatively from 0.3 mg/kg up to about20 mg/kg. In some applications, the dosage is from 0.5 mg/kg to 20mg/kg. In some instances, an antigen binding protein is dosed at 0.3mg/kg, 0.5 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg, or 20 mg/kg. The dosageschedule in some treatment regimes is at a dose of 0.3 mg/kg qW, 0.5mg/kg qW, 1 mg/kg qW, 3 mg/kg qW, 10 mg/kg qW, or 20 mg/kg qW.

Dosing frequency will depend upon the pharmacokinetic parameters of theparticular antigen binding protein in the formulation used. Typically, aclinician administers the composition until a dosage is reached thatachieves the desired effect. The composition can therefore beadministered as a single dose, or as two or more doses (which can butneed not contain the same amount of the desired molecule) over time, oras a continuous infusion via an implantation device or catheter.Appropriate dosages can be ascertained through use of appropriatedose-response data. In certain embodiments, the antigen binding proteinscan be administered to patients throughout an extended time period.Chronic administration of an antigen binding protein minimizes theadverse immune or allergic response commonly associated with antigenbinding proteins that are not fully human, for example an antibodyraised against a human antigen in a non-human animal, for example, anon-fully human antibody or non-human antibody produced in a non-humanspecies.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g., orally, through injection byintravenous, intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, or intralesional routes; by sustained release systems or byimplantation devices. In certain embodiments, the compositions can beadministered by bolus injection or continuously by infusion, or byimplantation device.

The composition also can be administered locally via implantation of amembrane, sponge or another appropriate material onto which the desiredmolecule has been absorbed or encapsulated. In certain embodiments,where an implantation device is used, the device can be implanted intoany suitable tissue or organ, and delivery of the desired molecule canbe via diffusion, timed-release bolus, or continuous administration.

It also can be desirable to use antigen binding protein pharmaceuticalcompositions ex vivo. In such instances, cells, tissues or organs thathave been removed from the patient are exposed to antigen bindingprotein pharmaceutical compositions after which the cells, tissuesand/or organs are subsequently implanted back into the patient.

In particular, antigen binding proteins that specifically bind (i)β-Klotho; (ii) FGFR1c, FGFR2c, FGFR3c or FGFR4; or (iii) a complexcomprising β-Klotho and one of FGFR1c, FGFR2c, FGFR3c, and FGFR4 can bedelivered by implanting certain cells that have been geneticallyengineered, using methods such as those described herein, to express andsecrete the polypeptide. In certain embodiments, such cells can beanimal or human cells, and can be autologous, heterologous, orxenogeneic. In certain embodiments, the cells can be immortalized. Inother embodiments, in order to decrease the chance of an immunologicalresponse, the cells can be encapsulated to avoid infiltration ofsurrounding tissues. In further embodiments, the encapsulation materialsare typically biocompatible, semi-permeable polymeric enclosures ormembranes that allow the release of the protein product(s) but preventthe destruction of the cells by the patient's immune system or by otherdetrimental factors from the surrounding tissues.

Combination Therapies

In another aspect, the present disclosure provides a method of treatinga subject for diabetes with a therapeutic antigen binding protein of thepresent disclosure, such as the fully human therapeutic antibodiesdescribed herein, together with one or more other treatments. In oneembodiment, such a combination therapy achieves an additive orsynergistic effect. The antigen binding proteins can be administered incombination with one or more of the type 2 diabetes or obesitytreatments currently available. These treatments for diabetes includebiguanide (metaformin), and sulfonylureas (such as glyburide,glipizide). Additional treatments directed at maintaining glucosehomeostasis include PPAR gamma agonists (pioglitazone, rosiglitazone);glinides (meglitinide, repaglinide, and nateglinide); DPP-4 inhibitors(Januvia® and Onglyza®) and alpha glucosidase inhibitors (acarbose,voglibose).

Additional combination treatments for diabetes include injectabletreatments such as insulin and incretin mimetics (Byetta®, Exenatide®),other GLP-1 (glucagon-like peptide) analogs such as liraglutide, otherGLP-1R agonists and Symlin® (pramlintide).

Additional combination treatments directed at weight loss includeMeridia® and Xenical®.

EXAMPLES

The following examples, including the experiments conducted and theresults achieved, are provided for illustrative purposes only and arenot to be construed as limiting.

Example 1 Preparation of FGFR1c Over Expressing Cells for Use as anAntigen

Nucleic acid sequences encoding the full length human FGFR1cpolypepetide (SEQ ID NO:4; FIGS. 1A-1B) and a separate sequence encodingthe full length human β-Klotho polypeptide (SEQ ID NO:7; FIGS. 2A-2C)were subcloned into suitable mammalian cell expression vectors (e.g.,pcDNA3.1 Zeo, pcDNA3.1 Hyg (Invitrogen, Carlsbad, Calif.) or pDSRα20.The pDSRα20 vector contains SV40 early promoter/enhancer for expressingthe gene of interest and a mouse DHFR expression cassette for selectionin CHO DHFR (−) host cells such as AM1 CHO (a derivative of DG44, CHODHFR (−)).

AM-1 CHO cells were seeded at 1.5×10⁶ cells per 100 mm dish. After 24hours, the cells were co-transfected with linearized DNAs ofpDSRα20/huFGFR1c and pDSRα20/huβ-Klotho with FuGene6 (Roche AppliedScience). The transfected cells were trypsinized 2 days aftertransfection and seeded into CHO DHFR selective growth medium containing10% dialyzed FBS and without hypoxanthine/thymidine supplement. After 2weeks, the resulting transfected colonies were trypsinized and pooled.

HEK293T cells were transfected with the full length huFGFR1c andhuβ-Klotho in pcDNA3.1 series or pTT14 (an expression vector developedby Durocher, NRCC, with CMV promoter and EBV ori, similar to pTT5 and apuromycin selection marker) based vector and selected with thecorresponding drugs following similar procedure as for the CHOtransfection and selection.

The FGF21R (i.e., FGFR1c and βKlotho) transfected AM1 CHO or 293T cellpools were sorted repeatedly using Alexa 647-labeled FGF21. As acell-surface staining reagent, FGF21 was labeled with Alexa 647-NHSfollowed the method recommended by the manufacturer (Molecular Probes,Inc. Cat A 2006). The Alexa 647-labeled FGF21 showed specific stainingof FGF21R receptor expressing cells and not the non-transfected parentalcells (FIG. 3). High expressing cells were collected at the end of thefinal sorting, expanded and frozen into vials. The AM-1/huFGF21R cellswere prepared for immunization and the 293T/huFGF21R cells were used fortitering mouse sera by FACS after immunization and in binding screens ofthe hybridoma supernatants by FMAT (see Example 4).

Example 2 Preparation of a Soluble FGFR1c/β-Klotho Complex for Use asAntigen

Soluble FGF21 receptor constructs were generated in pTT14 or pcDNA3.1expression vectors. The FGFR1c ECD-Fc construct (SEQ ID NO:362, FIG. 4)comprises the N-terminal extracellular domain of FGFR1c (amino acidresidues #1—374; SEQ ID NO:5) fused to Fc (SEQ ID NO:384). The β-KlothoECD-Fc construct (SEQ ID NO:363, FIG. 5) comprises the N-terminalextracellular domain of β-Klotho (amino acid residues #1-996; SEQ IDNO:8) fused to Fc (SEQ ID NO:384).

HEK293 cells (293F, Invitrogen) were transfected with huFGFR1cECD-Fc/pTT5, huβ-Klotho ECD-Fc/pTT14-puro and dGFP/pcDNA3.1-Neo andselected in the presence of the corresponding drugs followed by repeatedFACS sorting based on dGFP expression. Cells were grown in serum-freeDulbecco's Modified Eagle Medium (DMEM) supplemented with nonessentialamino acids in HyperFlasks (Corning) for 4 days and conditioned media(CM) harvested for purification.

The 293 CM was concentrated 6 fold and applied to Protein A FFequilibrated in PBS. The protein was eluted with Pierce Gentle Ag/Abelution buffer. The Protein A pool was dialyzed against 20 mM Tris-HCl,pH 7, 10 mM NaCl and applied to SP HP at pH 7.0. The FGFR1c ECD-Fc waspresent in the flow-through (FT) and the heterodimer was eluted withlinear gradient of 0-0.4 M NaCl, 20 mM Tris-HCl pH 7.0. N-terminus aminoacid sequencing verified the purified soluble FGF21R to be a heterodimercomposed of (1:1) ratio of FGFR1c ECD-Fc and β-Klotho ECD-Fc. Thepurified soluble FGF21R-Fc (FIG. 6) was used as the antigen forimmunization.

Example 3 Preparation of Monoclonal Antibodies

Immunizations were conducted using one or more suitable forms of FGF21receptor antigen, including: (1) cell bound receptor of CHOtransfectants expressing full length human FGFR1c and β-Klotho at thecell surface, obtained by transfecting CHO cells with cDNA encoding ahuman full length FGFR1c polypeptide of SEQ ID NO:4 (see also FIGS. 1a-b) and cDNA encoding a human β-Klotho polypeptide of SEQ ID NO:7 (seealso FIGS. 2 a-c); (2) membrane extract from the aforementioned cellsexpressing the FGF21R receptor complex; or (3) soluble FGF21R receptorobtainable by co-expressing the N-terminal extracellular domain (ECD) ofFGFR1c (SEQ ID NO:5; see also FIG. 4) and the N-terminal extracellulardomain (ECD) of β-Klotho (SEQ ID NO:8; see also FIG. 5) or (4)combinations thereof.

A suitable amount of immunogen (i.e., 10 μgs/mouse of soluble FGF21R or3−4×10⁶ cells/mouse of stably transfected CHO cells or 150 μgs/mouse ofpurified FGF21R membranes prepared from CHO cells stably expressingFGF21R) was used for initial immunization in XenoMouse™ according to themethods disclosed in U.S. patent application Ser. No. 08/759,620, filedDec. 3, 1996 and International Patent Application Nos. WO 98/24893, andWO 00/76310, the disclosures of which are hereby incorporated byreference. Following the initial immunization, subsequent boostimmunizations of immunogen (5 μg/mouse of soluble FGF21R or 1.7×10⁶FGF21R transfected cells/mouse or 75 μgs of purified FGF21R membranes)were administered on a schedule and for the duration necessary to inducea suitable anti-FGF21R titer in the mice. Titers were determined by asuitable method, for example, by enzyme immunoassay, fluorescenceactivated cell sorting (FACS), or by other methods (includingcombinations of enzyme immunoassays and FACS).

Animals exhibiting suitable titers were identified, and lymphocytes wereobtained from draining lymph nodes and, if necessary, pooled for eachcohort. Lymphocytes were dissociated from lymphoid tissue by grinding ina suitable medium (for example, Dulbecco's Modified Eagle Medium; DMEM;obtainable from Invitrogen, Carlsbad, Calif.) to release the cells fromthe tissues, and suspended in DMEM. B cells were selected and/orexpanded using standard methods, and fused with suitable fusion partner,for example, nonsecretory myeloma P3X63Ag8.653 cells (American TypeCulture Collection CRL1580; Kearney et al, J. Immunol. 123, 1979,1548-1550), using techniques that were known in the art.

In one suitable fusion method, lymphocytes were mixed with fusionpartner cells at a ratio of 1:4. The cell mixture was gently pelleted bycentrifugation at 400×g for 4 minutes, the supernatant decanted, and thecell mixture gently mixed (for example, by using a 1 ml pipette). Fusionwas induced with PEG/DMSO (polyethylene glycol/dimethyl sulfoxide;obtained from Sigma-Aldrich, St. Louis Mo.; 1 ml per million oflymphocytes). PEG/DMSO was slowly added with gentle agitation over oneminute followed, by one minute of mixing. IDMEM (DMEM without glutamine;2 ml per million of B cells), was then added over 2 minutes with gentleagitation, followed by additional IDMEM (8 ml per million B-cells) whichwas added over 3 minutes.

The fused cells were pelleted (400×g 6 minutes) and resuspended in 20 mlSelection media (for example, DMEM containing Azaserine and Hypoxanthine[HA] and other supplemental materials as necessary) per million B-cells.Cells were incubated for 20-30 minutes at 37° C. and then resuspended in200 ml selection media and cultured for three to four days in T175flasks prior to 96 well plating.

Cells were distributed into 96-well plates using standard techniques tomaximize clonality of the resulting colonies. After several days ofculture, supernatants were collected and subjected to screening assaysas detailed in the examples below, including confirmation of binding tohuman FGF21 receptor, specificity and/or cross-species reactivity.Positive cells were further selected and subjected to standard cloningand subcloning techniques. Clonal lines were expanded in vitro, and thesecreted human antibodies obtained for analysis.

In this manner, mice were immunized with either cells or membranesexpressing full length FGF21R cells, or soluble FGF21R extracellulardomain, with a range of 11-17 immunizations over a period ofapproximately one to three and one-half months. Several cell linessecreting FGF21R-specific antibodies were obtained, and the antibodieswere further characterized. The sequences thereof are presented hereinand in the Sequence Listing, and results of various tests using theseantibodies are provided.

Example 4 Selection of Binding Antibodies by FMAT

After 14 days of culture, hybridoma supernatants were screened forFGF21R-specific monoclonal antibodies by Fluorometric Microvolume AssayTechnology (FMAT) by screening against either the CHO AM1/huFGF21R cellline or recombinant HEK293 cells that were transfected with human FGF21Rand counter-screening against parental CHO or HEK293 cells. Briefly thecells in Freestyle media (Invitrogen) were seeded into 384-well FMATplates in a volume of 50 μL/well at a density of 4,000 cells/well forthe stable transfectants, and at a density of 16,000 cells/well for theparental cells, and cells were incubated overnight at 37° C. 10 μL/wellof supernatant was then added, and the plates were incubated forapproximately one hour at 4° C., after which 10 μL/well of anti-humanIgG-Cy5 secondary antibody was added at a concentration of 2.8 μg/ml(400 ng/ml final concentration). Plates were then incubated for one hourat 4° C., and fluorescence was read using an FMAT Cellular DetectionSystem (Applied Biosystems).

In total, over 3,000 hybridoma supernatants were identified as bindingto the FGF21 receptor expressing cells but not to parental cells by theFMAT method. These supernatants were then tested in the FGF21 functionalassays as described below.

Example 5 Selection of Antibodies that Induce FGF21-Like Signaling

Experiments were performed to identify functional antibodies that mimicwild-type FGF21 activity (e.g., the ability to induce FGF21-likesignaling) using a suitable FGF21 reporter assay. The disclosed FGF21reporter assay measures activation of FGFR signaling via a MAPK pathwayreadout. β-Klotho is a co-receptor for FGF21 signaling, and although itis believed not to have any inherent signaling capability due to itsvery short cytoplasmic domain, it is required for FGF21 to inducesignaling through FGFRs.

Example 5.1 ELK-Luciferase Reporter Assay

ELK-luciferase assays were performed using a recombinant human 293Tkidney cell or CHO cell system. Specifically, the host cells wereengineered to over-express β-Klotho and luciferase reporter constructs.The reporter constructs contain sequences encoding GAL4-ELK1 and5×UAS-Luc, a luciferase reporter driven by a promoter containing fivetandem copies of the Gal4 binding site. Activation of the FGF21 receptorcomplex in these recombinant reporter cell lines induces intracellularsignal transduction, which in turn leads to ERK and ELK phosphorylation.Luciferase activity is regulated by the level of phosphorylated ELK, andis used to indirectly monitor and quantify FGF21 activity.

In one example, CHO cells were transfected sequentially using theLipofectamine 2000 transfection reagent (Invitrogen) according to themanufacturer's protocol with the receptor constructs expressingβ-Klotho, FGFR1c and the reporter plasmids: 5× Gal4-Luciferase (minimalTK promoter with 5×Gal4 binding sites upstream of luciferase) andGal4-ELK1. Gal4-ELK1 binds to the Gal4 binding sites and activatestranscription when it is phosphorylated by ERK. Luciferasetranscription, and thereby the corresponding enzymatic activity in thiscontext is regulated by the level of phosphorylated ELK1, and is used toindirectly monitor and quantify FGF21 activity.

Clone 2E10 was selected as the FGF21 luciferase reporter cell line basedon the optimal assay window of 10-20 fold with native FGF21 exhibitingan EC50 in the single nM range.

For the assay, the ELK-luciferase reporter cells were plated in 96 wellassay plates, and serum starved overnight. FGF21 or test samples wereadded for 6 hours at 37 degrees. The plates were then allowed to cool toroom temperature and the luciferase activity in the cell lysates wasmeasured with Bright-Glo (Promega).

Example 5.2 ERK-Phosphorylation Assay

Alternative host cell lines specifically L6 (a rat myoblastic cell line)was developed and applied to identify antibodies with FGF21-likesignaling activity. The rat L6 cell line is a desirable host cell linefor the activity assay because it is known to express minimal levels ofendogeneous FGF receptors. The L6 cells do not respond to FGF21 evenwhen transfected with β-Klotho expression vector and therefore providesa cleaner background. (Kurosu et al., (2007) J. Biol. Chem. 282,26687-26695).

L6 cells were maintained in Dulbecco's modified Eagle's mediumsupplemented with 10% fetal bovine serum and penicillin/streptomycin.Cells were transfected with plasmids expressing βKlotho and individualFGFR using the Lipofectamine 2000 transfection reagent (Invitrogen)according to the manufacturer's protocol.

Analysis of FGF signaling in L6 cells was performed as described in theliterature (Kurosu et al., (2007) J. Biol. Chem. 282, 26687-26695). Cellcultures were collected 10 min after the treatment of FGF21 or testmolecules and snap frozen in liquid nitrogen, homogenized in the lysisbuffer and subjected to western blot analysis using ananti-phospho-p44/42 MAP kinase (ERK1/2) antibody and an anti-ERKantibody (Cell Signaling). The percent of phosphorylated ERK versustotal ERK protein was determined in this way.

In addition, the factor-dependent mouse BaF3 cell-based proliferationassay used frequently for cytokine receptors can also be developed andapplied.

Among the hybridoma supernatants tested in the CHO cell (clone 2E10)based human FGF21 ELK-luciferase reporter assay, over 30 were identifiedas positive (>5% of the activity of FGF21) when compared to 20 nM FGF21as the positive control. Antibodies were then purified from theconditioned media of the hybridoma cultures of these positives andtested again in the CHO cell based ELK-luciferase reporter assay. (FIG.7) showed the representative antibodies in the dose-responsive potencyassay with estimated EC50 less than 1 μg/ml (or 6.7 nM). The activitieswere confirmed in the L6 cell based ERK1/2-phosphrylation assay (FIG. 8)with EC50 less than 10 nM which is consistent to the ELK-luciferaseassay in the CHO stable cell line 2E10.

Example 6 Induction of FGF21-Like Signaling is Specific to theFGFR1c/βKlotho Complex

FGF21 has been reported to signal through multiple receptor complexesincluding FGFR1c, 2c, 3c and 4 when paired with β-Klotho. Theselectivity of the FGF21 agonistic antibodies was tested in the ratmyoblastic L6 cells transfected with vectors expressing the respectiveFGFRs and βKlotho. The results shown in FIG. 9 demonstrate that theactivity was mediated selectively and exclusively through FGFR1c and notthrough FGFR2c, 3c or 4 when they were paired with β-Klotho because noactivity was detected on the latter receptors up to 100 nM of theagonistic antibodies. This unique selectivity strongly suggests that theaction of these antibodies is β-Klotho-dependent yet it must alsoinvolve specifically the FGFR1c component of the signaling complex.

Example 7 Activity in Primary Human Adipocytes

FGF21 stimulates glucose uptake and lipolysis in cultured adipocytes,and adipocytes are considered to be more physiologically relevant thanthe recombinant reporter cell system.

A panel of the antibodies was shown to exhibit Erk-phosphorylationactivity similar to FGF21 in the human adipocyte assay (FIG. 10) withestimated EC50 less than 10 nM.

Example 8 Competition Binding and Epitope Binning

To compare the similarity of the binding sites of the antibodies on theFGF21 receptor, a series of competition binding experiments wereperformed and measured by Biacore™. In one example (and as shown in FIG.11), two representative agonistic FGF21 receptor antibodies (24H11 and17D8) and one non-functional FGF21 receptor binding antibodies (1A2.1)were immobilized on the sensor chip surface. Soluble humanFGFR1c/β-Klotho ECD-Fc complex or β-Klotho was then captured on theimmobilized antibody surfaces. Finally, several of the test FGF21receptor antibodies were injected individually over the captured solublehuman FGF21 receptor or β-Klotho. If the injected antibody recognizes adistinct binding site relative to that recognized by the immobilizedantibody, a second binding event will be observed. If the antibodiesrecognize very similar binding site, no more binding will be observed.

As shown in (FIG. 11A), there are two distinct yet partially overlappingbinding sites for the agonistic antibodies tested. One site is coveredby 24H11, 21H2, 18B11.1 and 17C3 (Group A) and the other site covered by17D8, 12E4 and 18G1 (Group B). The two non-functional antibodies 2G10and 1A2, bind to different sites from each other and are distinct fromthe two sites covered by the agonistic antibodies in Group A and B.Other functional antibodies binding to Group A epitope included 20D4,22H5, 16H7, 40D2 and 46D11. Two other functional antibodies 26H11 and37D3 were shown by this method to bind the same site covered by theGroup B antibodies. In addition, a third binding site for functionalantibodies was identified for 39F11, 39F7 and 39G5 (group C) whichappeared to be distinct from Group A and B binding sites (FIG. 11B).

Another Biacore™analysis was carried out with biotinylated-FGF21immobilized on the sensor ship. 10 nM soluble β-Klotho was then passedover the chip alone or mixed with the individual test antibodies at100nM. (FIG. 12) showed that several agonistic antibodies in group A(24H11, 18B11, 17C3) and antibody 12E4 (from group B) competedsignificantly with FGF21 in binding to soluble β-Klotho whereas thenon-functional antibodies 2G10 and 1A2 and several other functionalantibodies did not show competition binding with FGF21.

FIG. 11C summarizes the binning results obtained.

Example 9 Recognition of Native and Denatures Structures

The ability of disclosed antigen binding proteins to recognize denaturedand native structures was investigated. The procedure and results wereas follows.

Example 9.1 FGF21 Receptor Agonistic Antibodies do not RecognizeDenatured Structures, as Shown by FACS

Cell lysates from CHO cells stably expressing FGF21 receptor (FGFR1c andβ-Klotho) or CHO parental cells were diluted with sample buffer withoutbeta-mercaptoethanol (non-reducing conditions). 20 μl of cell lysate wasloaded per lane on adjacent lanes separated with a molecular weightmarker lane on 4-20% SDS-PAGE gels. Following electrophoresis, the gelswere blotted onto 0.2μ nitrocellulose filters. The blots were treatedwith Tris-buffered saline/Triton-X (TBST) plus 5% non-fat milk (blockingbuffer) for 30 minutes. The blots were then cut along the molecularweight marker lanes. The strips were then probed with FGF21 receptoragonistic antibodies (12C3, 26H11, 12E4, 21H2, 18B11, or 20D4), andcommercial goat anti-murine βKlotho or mouse anti-huFGFR1 (R&DDiagnostics) in TBST/5% milk. Blots were incubated with the antibodiesfor one hour at room temperature, followed by three washes with TBST+1%milk. The blots were then probed with anti-human or anti-goat IgG-HRPsecondary antibodies for 20 min. Blots were given three 15 min. washeswith TBST followed by treatment with Pierce Supersignal West Duradeveloping reagent (1 min.) and exposure to Kodak Biomax X-ray film.

The commercial anti-β-Klotho and anti-FGFR1 antibodies detected thecorresponding receptor proteins in the SDS-PAGE indicating they bind todenatured receptor proteins. In contrast, none of the FGF21 receptoragonistic antibodies tested detected the corresponding protein speciessuggesting they bind to the native conformational epitope distinct fromthe commercial antibodies which bind to denatured sequences.

Example 9.2 FGF21 Receptor Agonistic Antibodies Bind to Native ReceptorStructure, as Shown by FACS

A FACS binding assay was performed with several commercially availableFGFR1c and β-Klotho antibodies, and several of the disclosed FGF21receptor agonistic antibodies. The experiments were performed asfollows.

CHO cells stably expressing FGF21 receptor were treated with R&D Systemsmouse anti-huFGFR1, goat anti-mu β-Klotho, or FGF21 receptor antibodies24H11, 17C3, 17D8, 18G1, or 2G10 (1 μg per 1×10⁶ cells in 100 μlPBS/0.5% BSA). Cells were incubated with the antibodies at 4° C.followed by two washes with PBS/BSA. Cells were then treated withFITC-labeled secondary antibodies at 4° C. followed by two washes. Thecells were resuspended in 1 ml PBS/BSA and antibody binding was analyzedusing a FACS Calibur instrument.

Consistent with western blot results, all of the FGF21 receptoragonistic antibodies tested bind well to cell surface FGF21 receptor inFACS whereas the commercial anti-β-Klotho or anti-FGFR1 antibodies didnot. This observation further confirmed that the FGF21 receptoragonistic antibodies recognize the native structure whereas thecommercial antibodies to the receptor components do not.

Example 10 Arginine Scanning

As described above, antigen binding proteins that bind human FGF21R,e.g., FGFR1c, β-Klotho or both FGFR1c and β-Klotho, were created andcharacterized. To determine the neutralizing determinants on humanFGFR1c and/or β-Klotho that these various antigen binding proteinsbound, a number of mutant FGFR1c and/or β-Klotho proteins can beconstructed having arginine substitutions at select amino acid residuesof human FGFR1c and/or β-Klotho. Arginine scanning is an art-recognizedmethod of evaluating where antibodies, or other proteins, bind toanother protein, see, e.g., Nanevicz et al., (1995) J. Biol. Chem.,270:37, 21619-21625 and Zupnick et al., (2006) J. Biol. Chem., 281:29,20464-20473. In general, the arginine sidechain is positively chargedand relatively bulky as compared to other amino acids, which can disruptantibody binding to a region of the antigen where the mutation isintroduced. Arginine scanning is a method that determines if a residueis part of a neutralizing determinant and/or an epitope.

Various amino acids distributed throughout the human FGFR1c and/orβ-Klotho extracellular domains can be selected for mutation to arginine.The selection can be biased towards charged or polar amino acids tomaximize the possibility of the residue being on the surface and reducethe likelihood of the mutation resulting in misfolded protein. Usingstandard techniques known in the art, sense and anti-senseoligonucleotides containing the mutated residues can be designed basedon criteria provided by Stratagene Quickchange® II protocol kit(Stratagene/Agilent, Santa Clara, Calif.). Mutagenesis of the wild-type(WT) FGFR1c and/or β-Klotho sequences can be performed using aQuickchange® II kit (Stratagene). Chimeric constructs can be engineeredto encode a FLAG-histidine tag (six histidines (SEQ ID NO: 382)) on thecarboxy terminus of the extracellular domain to facilitate purificationvia the poly-His tag.

Multiplex analysis using the Bio-Plex®Workstation and software(Bio-Rad®, Hercules, Calif.) can be performed to determine neutralizingdeterminants on human FGFR1c and/or β-Klotho by analyzing exemplaryhuman FGFR1c and/or β-Klotho mAbs differential binding toargininemutants versus wild-type FGFR1c and/or β-Klotho proteins. Anynumber of bead codes of pentaHis-coated beads (“penta-His” disclosed asSEQ ID NO: 383) (Qiagen®, Valencia, Calif.) can be used to capturehistidine-tagged protein. The bead codes can allow the multiplexing ofFGFR1c and/or β-Klotho arginine mutants and wild-type human FGFR1cand/or β-Klotho.

To prepare the beads, 100 ul of wild-type FGFR1c and/or β-Klotho andFGFR1c and/or β-Klotho arginine mutant supernatants from transientexpression culture are bound to penta-His-coated beads (“penta-His”disclosed as SEQ ID NO: 383) overnight at 4° C. or 2 hours at roomtemperature with vigorous shaking. The beads are then washed as per themanufacturer's protocol and the bead set pooled and aliquoted into 2 or3 columns of a 96-well filter plate (Millipore, Bellerica, Mass.,product #MSBVN1250) for duplicate or triplicate assay points,respectively. 100 μl anti-FGFR1c and/or anti-β-Klotho antibodies in4-fold dilutions are added to the wells, incubated for 1 hour at roomtemperature, and washed. 100 μl of a 1:100 dilution of PE-conjugatedanti-human IgG Fc (Jackson Labs., Bar Harbor, Me., product #109-116-170)is added to each well, incubated for 1 hour at room temperature andwashed. Beads are resuspended in 1% BSA, shaken for 3 minutes, and readon the Bio-Plex workstation. Antibody binding to FGFR1c and/or β-Klothoarginine mutant protein is compared to antibody binding to the humanFGFR1c and/or β-Klotho wild-type from the same pool. A titration ofantibody over approximately a 5 log scale can be performed. MedianFluorescence Intensity (MFI) of FGFR1c and/or β-Klotho arginine mutantproteins can be graphed as a percent of maximum wild-type human FGFR1cand/or β-Klotho signal. Those mutants for which signal from all theantibodies are below a cut-off value, e.g., 30% of wild-type FGFR1cand/or β-Klotho can be deemed to be either of too low a proteinconcentration on the bead due to poor expression in the transientculture or possibly misfolded and can be excluded from analysis.Mutations (i.e., arginine substitutions) that increase the EC50 for theFGFR1c and/or β-Klotho mAb by a cut-off value, e.g., 3-fold or greater(as calculated by, e.g., GraphPad Prism®) can be considered to havenegatively affected FGFR1c and/or β-Klotho mAb binding. Through thesemethods, neutralizing determinants and epitopes for various FGFR1cand/or β-Klotho antibodies are elucidated.

Example 11 Construction of Chimeric Receptors

In another method of determining the activation determinants on humanFGFR1c and/or β-Klotho that these various antigen binding proteins bind,specific chimeric FGFR1c and/or β-Klotho proteins between human andmouse species can be constructed, expressed in transient or stable 293or CHO cells as described before and tested. For example, a chimericFGF21 receptor can be constructed comprising native human FGFR1c,FGFR2c, FGFR3c or FGFR4, in one example FGFR1c, paired with chimerichuman/mouse β-Klotho in which selected regions or sequences on the humanβ-Klotho are systematically replaced by the corresponding mouse-specificresidues (see, e.g., FIG. 2A-2C). Similarly, native human β-Klothopaired with chimeric human/mouse FGFR1c, FGFR2c, FGFR3c or FGFR4, in oneexample FGFR1c in which selected regions or sequences on the humanFGFR1c are systematically replaced by the corresponding mouse-specificresidues (see, e.g., the alignments of FIGS. 1A-1B). The criticalsequences involved in the binding and/or activity of the antigen bindingproteins can be derived through binding assay or activity measurementsdescribed in previous Examples 4, 5, 6 and 7 based on the chimeric FGF21receptors.

Example 11.1 Construction of Specific Chimeras

Human-mouse β-Klotho chimeras were constructed using the methodologydescribed in Example 14. A schematic of the chimeras constructed ispresented in FIG. 29; summarily, the chimeras generated comprised (fromN to C terminus) a fusion of a human β-Klotho sequence fused to a murineβ-Klotho sequence fused to a human β-Klotho sequence. Human β-Klotho(SEQ ID NO:8) was used as a framework into which regions of murineβ-Klotho (full length sequence shown in SEQ ID NO:468) were inserted.The regions of murine β-Klotho that were inserted were as follows:

Murine Residues 82P-520P (SEQ ID NO: 470)PKNFSWGVGTGAFQVEGSWKTDGRGPSIWDRYVYSHLRGVNGTDRSTDSYIFLEKDLLALDFLGVSFYQFSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEPIVTLYHWDLPLTLQEEYGGWKNATMIDLFNDYATYCFQTFGDRVKYWITIHNPYLVAWHGFGTGMHAPGEKGNLTAVYTVGHNLIKAHSKVWHNYDKNFRPHQKGWLSITLGSHWIEPNRTDNMEDVINCQHSMSSVLGWFANPIHGDGDYPEFMKTGAMIPEFSEAEKEEVRGTADFFAFSFGPNNFRPSNTVVKMGQNVSLNLRQVLNWIKLEYDDPQILISENGWFTDSYIKTEDTTAIYMMKNFLNQVLQAIKFDEIRVFGYTAWTLLDGFEWQDAYTTRRGLFYVDFNSEQKERKPKSSAHYYKQIIQDNGFPLKESTPDMKGRFP Murine Residues 506F-1043S (SEQ ID NO:471) FPLKESTPDMKGRFPCDFSWGVTESVLKPEFTVSSPQFTDPHLYVWNVTGNRLLYRVEGVRLKTRPSQCTDYVSIKKRVEMLAKMKVTHYQFALDWTSILPTGNLSKVNRQVLRYYRCVVSEGLKLGVFPMVTLYHPTHSHLGLPLPLLSSGGWLNMNTAKAFQDYAELCFRELGDLVKLWITINEPNRLSDMYNRTSNDTYRAAHNLMIAHAQVWHLYDRQYRPVQHGAVSLSLHCDWAEPANPFVDSHWKAAERFLQFEIAWFADPLFKTGDYPSVMKEYIASKNQRGLSSSVLPRFTAKESRLVKGTVDFYALNHFTTRFVIHKQLNTNRSVADRDVQFLQDITRLSSPSRLAVTPWGVRKLLAWIRRNYRDRDIYITANGIDDLALEDDQIRKYYLEKYVQEALKAYLIDKVKIKGYYAFKLTEEKSKPRFGFFTSDFRAKSSVQFYSKLISSSGLPAENRSPACGQPAEDTDCTICSFLVEKKPLIFFGCCFISTLAVLLSITVFHHQKRRKFQKARNLQNIPLKK GHSRVFSMurine Residues 1M-193L (SEQ ID NO: 472)MKTGCAAGSPGNEWIFFSSDERNTRSRKTMSNRALQRSAVLSAFVLLRAVTGFSGDGKAIWDKKQYVSPVNPSQLFLYDTFPKNFSWGVGTGAFQVEGSWKTDGRGPSIWDRYVYSHLRGVNGTDRSTDSYIFLEKDLLALDFLGVSFYQFSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEPIVTL Murine Residues 82P-302S (SEQ ID NO: 473)PKNFSWGVGTGAFQVEGSWKTDGRGPSIWDRYVYSHLRGVNGTDRSTDSYIFLEKDLLALDFLGVSFYQFSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEPIVTLYHWDLPLTLQEEYGGWKNATMIDLFNDYATYCFQTFGDRVKYWITIHNPYLVAWHGFGTGMHAPGEKGNLTAVYTVGHNLIKAHSKVWHNYDKNFRPHQKGWLSITLGS Murine Residues 194Y-416G(SEQ ID NO: 474)YHWDLPLTLQEEYGGWKNATMIDLFNDYATYCFQTFGDRVKYWITIHNPYLVAWHGFGTGMHAPGEKGNLTAVYTVGHNLIKAHSKVWHNYDKNFRPHQKGWLSITLGSHWIEPNRTDNMEDVINCQHSMSSVLGWFANPIHGDGDYPEFMKTGAMIPEFSEAEKEEVRGTADFFAFSFGPNNFRPSNTVVKMGQNVSLNLRQVLNWIKLEYDDPQILISENG Murine Residues302S-506F (SEQ ID NO: 475)SHWIEPNRTDNMEDVINCQHSMSSVLGWFANPIHGDGDYPEFMKTGAMIPEFSEAEKEEVRGTADFFAFSFGPNNFRPSNTVVKMGQNVSLNLRQVLNWIKLEYDDPQILISENGWFTDSYIKTEDTTAIYMMKNFLNQVLQAIKFDEIRVFGYTAWTLLDGFEWQDAYTTRRGLFYVDFNSEQKERKPKSSAHYYKQIIQDNGF Murine Residues 416G-519P (SEQ ID NO: 476)GWFTDSYIKTEDTTAIYMMKNFLNQVLQAIKFDEIRVFGYTAWTLLDGFEWQDAYTTRRGLFYVDFNSEQKERKPKSSAHYYKQIIQDNGFPLKESTPDMKGRF Murine Residues 507P-632G(SEQ ID NO: 477)PLKESTPDMKGRFPCDFSWGVTESVLKPEFTVSSPQFTDPHLYVWNVTGNRLLYRVEGVRLKTRPSQCTDYVSIKKRVEMLAKMKVTHYQFALDWTSILPTGNLSKVNRQVLRYYRC VVSEGLKLGMurine Residues 520P-735A (SEQ ID NO: 478)PCDFSWGVTESVLKPEFTVSSPQFTDPHLYVWNVTGNRLLYRVEGVRLKTRPSQCTDYVSIKKRVEMLAKMKVTHYQFALDWTSILPTGNLSKVNRQVLRYYRCVVSEGLKLGVFPMVTLYHPTHSHLGLPLPLLSSGGWLNMNTAKAFQDYAELCFRELGDLVKLWITINEPNRLSDMYNRTSNDTYRAAHNLMIAHAQVWHLYDRQYRPVQHGA Murine Residues 632G-849Q (SEQID NO: 479) GVFPMVTLYHPTHSHLGLPLPLLSSGGWLNMNTAKAFQDYAELCFRELGDLVKLWITINEPNRLSDMYNRTSNDTYRAAHNLMIAHAQVWHLYDRQYRPVQHGAVSLSLHCDWAEPANPFVDSHWKAAERFLQFEIAWFADPLFKTGDYPSVMKEYIASKNQRGLSSSVLPRFTAKESRLVKGTVDFYALNHFTTRFVIHKQLNTNRSVADRDVQFLQ Murine Residues 735A-963S(SEQ ID NO: 480)AVSLSLHCDWAEPANPFVDSHWKAAERFLQFEIAWFADPLFKTGDYPSVMKEYIASKNQRGLSSSVLPRFTAKESRLVKGTVDFYALNHFTTRFVIHKQLNTNRSVADRDVQFLQDITRLSSPSRLAVTPWGVRKLLAWIRRNYRDRDIYITANGIDDLALEDDQIRKYYLEKYVQEALKAYLIDKVKIKGYYAFKLTEEKSKPRFGFFTSDFRAKSSVQFYSKLISSS Murine Residues1M-81F (SEQ ID NO: 481)MKTGCAAGSPGNEWIFFSSDERNTRSRKTMSNRALQRSAVLSAFVLLRAVTGFSGDGKAIWDKKQYVSPVNPSQLFLYDTF Murine Residues 82P-193L (SEQ ID NO: 482)PKNFSWGVGTGAFQVEGSWKTDGRGPSIWDRYVYSHLRGVNGTDRSTDSYIFLEKDLLALDFLGVSFYQFSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEPIVTL

The chimeras generated using the murine β-Klotho sequences comprised thefollowing components:

Construct N-terminal C-terminal SEQ ID Human β- Mouse β-Klotho Humanβ-Klotho Construct Identifier NO Klotho Residues Residues ResidueshuBeta_Klotho(1-81, 523-1044) 1-81   82-520 523-1044 (muBetaKLOTHO82-520) huBeta_Klotho(1-507) 1-507  506-1043 (muBetaKLOTHO 506F-1045S)huBeta_Klotho(194-1044)  1-193 194-1044 (muBetaKLOTHO 1-L193)huBeta_Klotho(1-81, 303-1044) 1-81   82-302 303-1044 (muBetaKLOTHO82P-302S) huBeta_Klotho(1-193, 419-1044) 1-193 194-416 419-1044(muBetaKLOTHO Y194-416G) huBeta_Klotho(1-301, 509-1044) 1-301 302-506509-1044 (muBetaKLOTHO S302-F506) huBeta_Klotho(1-417, 522-1044) 1-417416-519 522-1044 (muBetaKLOTHO G416-F519) huBeta_Klotho(1-507, 635-1044)1-508 507-632 635-1044 (muBeta KLOTHO F06-G632) huBeta_Klotho(1-521,738-1044) 1-521 520-735 738-1044 (muBeta KLOTHO 520P-735A)huBeta_Klotho(1-633, 852-1044) 1-633 632-849 852-1044 (muBeta KLOTHO632G-849Q) huBeta_Klotho(1-736, 967-1044) 1-736 735-963 967-1044 (muBetaKLOTHO 735A-963S) huBeta_Klotho(82-1044)(muBeta  1-81  82-1044 KLOTHO1-81F) huBeta_Klotho(1-81, 194-1044) 1-81   82-193 194-1044 (muBetaKLOTHO 82P-193L)

The generated chimeras comprised the following amino acid sequences:

(i) huBeta_Klotho(1-81, 523-1044)(muBetaKLOTHO 82-520) (SEQ ID NO: 455)MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAVTGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFSWGVGTGAFQVEGSWKTDGRGPSIWDRYVYSHLRGVNGTDRSTDSYIFLEKDLLALDFLGVSFYQFSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEPIVTLYHWDLPLTLQEEYGGWKNATMIDLFNDYATYCFQTFGDRVKYWITIHNPYLVAWHGFGTGMHAPGEKGNLTAVYTVGHNLIKAHSKVWHNYDKNFRPHQKGWLSITLGSHWIEPNRTDNMEDVINCQHSMSSVLGWFANPIHGDGDYPEFMKTGAMIPEFSEAEKEEVRGTADFFAFSFGPNNFRPSNTVVKMGQNVSLNLRQVLNWIKLEYDDPQILISENGWFTDSYIKTEDTTAIYMMKNFLNQVLQAIKFDEIRVFGYTAWTLLDGFEWQDAYTTRRGLFYVDFNSEQKERKPKSSAHYYKQIIQDNGFPLKESTPDMKGRFPCDFSWGVTESVLKPESVASSPQFSDPHLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLGLPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYNRSGNDTYGAAHNLLVAHALAWRLYDQQFRPSQRGAVSLSLHADWAEPANPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFLGCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRVVS (ii)huBeta_Klotho(1-507)(muBetaKLOTHO 506F-1045S) (SEQ ID NO: 456)MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAVTGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSWKKDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQFSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPLALQEKYGGWKNDTIIDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGYGTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITLGSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFSVLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREALNWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLDEIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYKQIIRENGFPLKESTPDMKGRFPCDFSWGVTESVLKPEFTVSSPQFTDPHLYVWNVTGNRLLYRVEGVRLKTRPSQCTDYVSIKKRVEMLAKMKVTHYQFALDWTSILPTGNLSKVNRQVLRYYRCVVSEGLKLGVFPMVTLYHPTHSHLGLPLPLLSSGGWLNMNTAKAFQDYAELCFRELGDLVKLWITINEPNRLSDMYNRTSNDTYRAAHNLMIAHAQVWHLYDRQYRPVQHGAVSLSLHCDWAEPANPFVDSHWKAAERFLQFEIAWFADPLFKTGDYPSVMKEYIASKNQRGLSSSVLPRFTAKESRLVKGTVDFYALNHFTTRFVIHKQLNTNRSVADRDVQFLQDITRLSSPSRLAVTPWGVRKLLAWIRRNYRDRDIYITANGIDDLALEDDQIRKYYLEKYVQEALKAYLIDKVKIKGYYAFKLTEEKSKPRFGFFTSDFRAKSSVQFYSKLISSSGLPAENRSPACGQPAEDTDCTICSFLVEKKPLIFFGCCFISTLAVLLSITVFHHQKRRKFQKARNLQNIPLKKGHSRVFS (iii)huBeta_Klotho(194-1044)(muBetaKLOTHO 1-L193) (SEQ ID NO: 457)MKTGCAAGSPGNEWIFFSSDERNTRSRKTMSNRALQRSAVLSAFVLLRAVTGFSGDGKAIWDKKQYVSPVNPSQLFLYDTFPKNFSWGVGTGAFQVEGSWKTDGRGPSIWDRYVYSHLRGVNGTDRSTDSYIFLEKDLLALDFLGVSFYQFSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEPIVTLYHWDLPLALQEKYGGWKNDTIIDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGYGTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITLGSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFSVLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREALNWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLDEIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYKQIIRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLGLPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYNRSGNDTYGAAHNLLVAHALAWRLYDQQFRPSQRGAVSLSLHADWAEPANPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFLGCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRVVS (iv) huBeta_Klotho(1-81,303-1044)(muBetaKLOTHO 82P-302S) (SEQ ID NO: 458)MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAVTGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFSWGVGTGAFQVEGSWKTDGRGPSIWDRYVYSHLRGVNGTDRSTDSYIFLEKDLLALDFLGVSFYQFSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEPIVTLYHWDLPLTLQEEYGGWKNATMIDLFNDYATYCFQTFGDRVKYWITIHNPYLVAWHGFGTGMHAPGEKGNLTAVYTVGHNLIKAHSKVWHNYDKNFRPHQKGWLSITLGSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFSVLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREALNWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLDEIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYKQIIRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLGLPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYNRSGNDTYGAAHNLLVAHALAWRLYDQQFRPSQRGAVSLSLHADWAEPANPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFLGCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRVVS (v) huBeta_Klotho(1-193,419-1044)(muBetaKLOTHO Y194-416G) (SEQ ID NO: 459)MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAVTGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSWKKDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQFSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPLTLQEEYGGWKNATMIDLFNDYATYCFQTFGDRVKYWITIHNPYLVAWHGFGTGMHAPGEKGNLTAVYTVGHNLIKAHSKVWHNYDKNFRPHQKGWLSITLGSHWIEPNRTDNMEDVINCQHSMSSVLGWFANPIHGDGDYPEFMKTGAMIPEFSEAEKEEVRGTADFFAFSFGPNNFRPSNTVVKMGQNVSLNLRQVLNWIKLEYDDPQILISENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLDEIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYKQIIRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLGLPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYNRSGNDTYGAAHNLLVAHALAWRLYDQQFRPSQRGAVSLSLHADWAEPANPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFLGCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRVVS (vi) huBeta_Klotho(1-301,509-1044)(muBetaKLOTHO S302-F506) (SEQ ID NO: 460)MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAVTGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSWKKDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQFSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPLALQEKYGGWKNDTIIDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGYGTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITLGSHWIEPNRTDNMEDVINCQHSMSSVLGWFANPIHGDGDYPEFMKTGAMIPEFSEAEKEEVRGTADFFAFSFGPNNFRPSNTVVKMGQNVSLNLRQVLNWIKLEYDDPQILISENGWFTDSYIKTEDTTAIYMMKNFLNQVLQAIKFDEIRVFGYTAWTLLDGFEWQDAYTTRRGLFYVDFNSEQKERKPKSSAHYYKQIIQDNGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLGLPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYNRSGNDTYGAAHNLLVAHALAWRLYDQQFRPSQRGAVSLSLHADWAEPANPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFLGCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRVVS (vii) huBeta_Klotho(1-417,522-1044)(muBetaKLOTHO G416-F519) (SEQ ID NO: 461)MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAVTGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSWKKDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQFSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPLALQEKYGGWKNDTIIDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGYGTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITLGSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFSVLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREALNWIKLEYNNPRILIAENGWFTDSYIKTEDTTAIYMMKNFLNQVLQAIKFDEIRVFGYTAWTLLDGFEWQDAYTTRRGLFYVDFNSEQKERKPKSSAHYYKQIIQDNGFPLKESTPDMKGRFPCDFSWGVTESVLKPESVASSPQFSDPHLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLGLPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYNRSGNDTYGAAHNLLVAHALAWRLYDQQFRPSQRGAVSLSLHADWAEPANPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFLGCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRVVS (viii) huBeta_Klotho(1-507,635-1044)(muBeta KLOTHO F06-G632) (SEQ ID NO: 462)MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAVTGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSWKKDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQFSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPLALQEKYGGWKNDTIIDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGYGTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITLGSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFSVLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREALNWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLDEIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYKQIIRENGFPLKESTPDMKGRFPCDFSWGVTESVLKPEFTVSSPQFTDPHLYVWNVTGNRLLYRVEGVRLKTRPSQCTDYVSIKKRVEMLAKMKVTHYQFALDWTSILPTGNLSKVNRQVLRYYRCVVSEGLKLGISAMVTLYYPTHAHLGLPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYNRSGNDTYGAAHNLLVAHALAWRLYDQQFRPSQRGAVSLSLHADWAEPANPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFLGCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRVVS (ix) huBeta_Klotho(1-521,738-1044)(muBeta KLOTHO 520P-735A) (SEQ ID NO: 463)MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAVTGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSWKKDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQFSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPLALQEKYGGWKNDTIIDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGYGTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITLGSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFSVLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREALNWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLDEIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYKQIIRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPEFTVSSPQFTDPHLYVWNVTGNRLLYRVEGVRLKTRPSQCTDYVSIKKRVEMLAKMKVTHYQFALDWTSILPTGNLSKVNRQVLRYYRCVVSEGLKLGVFPMVTLYHPTHSHLGLPLPLLSSGGWLNMNTAKAFQDYAELCFRELGDLVKLWITINEPNRLSDMYNRTSNDTYRAAHNLMIAHAQVWHLYDRQYRPVQHGAVSLSLHADWAEPANPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFLGCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRVVS (x) huBeta_Klotho(1-633,852-1044)(muBeta KLOTHO 632G-849Q) (SEQ ID NO: 464)MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAVTGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSWKKDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQFSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPLALQEKYGGWKNDTIIDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGYGTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITLGSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFSVLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREALNWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLDEIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYKQIIRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGVFPMVTLYHPTHSHLGLPLPLLSSGGWLNMNTAKAFQDYAELCFRELGDLVKLWITINEPNRLSDMYNRTSNDTYRAAHNLMIAHAQVWHLYDRQYRPVQHGAVSLSLHCDWAEPANPFVDSHWKAAERFLQFEIAWFADPLFKTGDYPSVMKEYIASKNQRGLSSSVLPRFTAKESRLVKGTVDFYALNHFTTRFVIHKQLNTNRSVADRDVQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFLGCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRVVS (xi) huBeta_Klotho(1-736,967-1044)(muBeta KLOTHO 735A-963S) (SEQ ID NO: 465)MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAVTGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSWKKDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQFSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPLALQEKYGGWKNDTIIDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGYGTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITLGSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFSVLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREALNWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLDEIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYKQIIRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLGLPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYNRSGNDTYGAAHNLLVAHALAWRLYDQQFRPSQRGAVSLSLHCDWAEPANPFVDSHWKAAERFLQFEIAWFADPLFKTGDYPSVMKEYIASKNQRGLSSSVLPRFTAKESRLVKGTVDFYALNHFTTRFVIHKQLNTNRSVADRDVQFLQDITRLSSPSRLAVTPWGVRKLLAWIRRNYRDRDIYITANGIDDLALEDDQIRKYYLEKYVQEALKAYLIDKVKIKGYYAFKLTEEKSKPRFGFFTSDFRAKSSVQFYSKLISSSGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFLGCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRVVS (xii)huBeta_Klotho(82-1044)(muBeta KLOTHO 1-81F) (SEQ ID NO: 466)MKTGCAAGSPGNEWIFFSSDERNTRSRKTMSNRALQRSAVLSAFVLLRAVTGFSGDGKAIWDKKQYVSPVNPSQLFLYDTFPKNFFWGIGTGALQVEGSWKKDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQFSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPLALQEKYGGWKNDTIIDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGYGTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITLGSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFSVLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREALNWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLDEIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYKQIIRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLGLPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYNRSGNDTYGAAHNLLVAHALAWRLYDQQFRPSQRGAVSLSLHADWAEPANPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFLGCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRVVS (xiii) huBeta_Klotho(1-81,194-1044)(muBeta KLOTHO 82P-193L) (SEQ ID NO: 467)MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAVTGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFSWGVGTGAFQVEGSWKTDGRGPSIWDRYVYSHLRGVNGTDRSTDSYIFLEKDLLALDFLGVSFYQFSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEPIVTLYHWDLPLALQEKYGGWKNDTIIDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGYGTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITLGSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFSVLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREALNWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLDEIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYKQIIRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLGLPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYNRSGNDTYGAAHNLLVAHALAWRLYDQQFRPSQRGAVSLSLHADWAEPANPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFLGCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRVVS

Various antigen binding proteins provided herein, as well as humanFGF21, were tested for the ability to activate the chimeras in L6 cells.FIG. 30 correlates the observed results with each tested molecule.

These data indicate that while human FGF21 was able to activate FGFR1ccombined with all of the human/mouse β-Klotho chimeras (“+” signindicate activity on the receptor), the substitutions of mouse sequencesinto human β-Klotho affected the activities of 16H7, 37D3, and 39F7. SeeFIG. 30. These results suggest that β-Klotho sequences 1-81, 302-522,and 849-1044 are important for the activities of agonistic antigenbinding proteins and may represent an important epitope for theirfunction.

Example 12 Protease Protection Analysis

Regions of the human FGF21 receptor bound by the antigen bindingproteins that bind human FGF21 receptor, e.g., FGFR1c, β-Klotho orFGFR1c and β-Klotho complex can be identified by fragmenting human FGF21receptor into peptides with specific proteases, e.g., AspN, Lys-C,chymotrypsin or trypsin. The sequence of the resulting human FGF21receptor peptides (i.e., both disulfide- and non-disulfide-containingpeptide fragments from FGFR1c and β-Klotho portions) can then bedetermined. In one example, soluble forms of a human FGF21 receptor,e.g., a complex comprising the FGFR1c ECD-Fc and β-Klotho ECD-Fcheterodimer described herein can be digested with AspN (which cleavesafter aspartic acid and some glutamic acid residues at the amino end) byincubating about 100 μg of soluble FGF21 receptor at 1.0 mg/ml in 0.1Msodium phosphate (pH 6.5) for 20 hrs at 37° C. with 2 μg of AspN.

A peptide profile of the AspN digests can then be generated on HPLCchromatography while a control digestion with a similar amount ofantibody is expected to be essentially resistant to AspN endoprotease. Aprotease protection assay can then be performed to determine theproteolytic digestion of human FGF21 receptor in the presence of theantigen binding proteins. The general principle of this assay is thatbinding of an antigen binding protein to the FGF21 receptor can resultin protection of certain specific protease cleavage sites and thisinformation can be used to determine the region or portion of FGF21receptor where the antigen binding protein binds.

Briefly, the peptide digests can be subjected to HPLC peptide mapping;the individual peaks are collected, and the peptides are identified andmapped by on-line electrospray ionization LC-MS (ESI-LC-MS) analysesand/or by N-terminal sequencing. HPLC analyses for these studies can beperformed using a narrow bore reverse-phase C18 column (AgilentTechnologies) for off-line analysis and using a capillary reverse phaseC18 column (The Separation Group) for LC-MS. HPLC peptide mapping can beperformed with a linear gradient from 0.05% trifluoroacetic acid (mobilephase A) to 90% acetonitrile in 0.05% trifluoroacetic acid. Columns canbe developed at desirable flow rate for narrow bore HPLC for off-line oron-line LC-MS analyses, and for capillary HPLC for on-line LC-MSanalyses.

Sequence analyses can be conducted by on-line LC-MS/MS and by Edmansequencing on the peptide peaks recovered from HPLC. On-line ESI LC-MSanalyses of the peptide digest can be performed to determine the precisemass and sequence of the peptides that are separated by HPLC. Theidentities of selected peptides present in the peptide peaks from theprotease digestion can thus be determined.

Example 13 Cynomolgous Monkey Study

A construct encoding the antigen binding protein designated herein as16H7 was generated using the methodology disclosed in Examples 1-3. 16H7was expressed, purified and characterized as described in Examples 1-5and was studied in vivo in obese cynomolgus monkeys. 16H7 is a fullyhuman IgG1 antibody and is described by the sequences provided in Tables1-4, supra.

Example 13.1 Study Design

The study was conducted in obese cynomolgus monkeys. The monkeys were8-19 years old. Their body weights ranged from 7-14 kg and BMI rangedfrom 36-74 kg/m². Monkeys were acclimated for 6 weeks prior to theinitiation of compound administration. During the acclimation period,the monkeys were familiarized with study-related procedures, includingchair-restraint, subcutaneous injection (PBS, 0.1 ml/kg), gavage (water,10 ml/kg), and blood drawn for non-OGTT and OGTT samples. After 4 weeksof training, baseline OGTT and plasma metabolic parameters weremeasured. 20 monkeys were selected and randomized into two treatmentgroups to achieve similar baseline levels of body weight, glucose OGTTprofiles, and plasma glucose and triglyceride levels.

The study was conducted in a blinded fashion. Vehicle (n=10), 16H7(n=10). Compound was given every other week (5 mg/kg). On the week whenanimals were not injected with 16H7, they received vehicle injectioninstead. After 2 injections of 16H7, animals were monitored during anadditional 6 weeks for compound washout and recovery from treatments.Food intake, body weight, clinical chemistry and OGTT were monitoredthroughout the study. Food intake was measured every meal. Body weightwas measured weekly. Blood samples were collected on different days infasted or fed state to measure glucose, insulin and triglyceride levels.OGTTs were conducted every two weeks after the initiation of the study.The day starting the treatment is designated as 0 and the detailed studyplan is shown in FIG. 14.

The results presented in this Example represent data collectedthroughout the 68 days of the study.

Example 13.2 Effect of 16H7 on Food Intake

Animals were fed twice a day, with each animal receiving 120 g offormulated food established during the acclimation period. The remainingfood was removed and weighed after each meal to calculate food intake.The feeding times were from 8:00 AM to 8:30 AM (±30 minutes) and thenfrom 4:30 PM to 5:00 PM (±30 minutes). Fruit (150 g) was supplied toeach animal at 11:30 to 12:30 PM (±30 minutes) every day.

Compared with vehicle, 16H7 reduced food intake in the monkeys. Theeffect diminished and the food intake returned to close to baseline orcontrol levels after about 21 days of treatment. 16H7 did not have asignificant effect on AM food intake (FIG. 15) and only modestly reducedfood intake on PM meal during the treatment (FIG. 16). An increase in AMfood intake was seen after day 49 (FIG. 15). Throughout the study (andeven during the acclimation period), fruit intake seemed lower in the16H7 group compared to the vehicle group. Overall, 16H7 showed asignificant effect on inhibiting food intake.

Example 13.3 Effect of 16H7 on Body Weight

Body weight was monitored weekly throughout the study. Over the courseof the 4 week treatments, the body weight of animals treated withvehicle remained constant while body weight of animals treated with 16H7progressively decreased. Body weight did not return to baseline by theend of the 6 weeks wash out period (FIG. 17).

Example 13.4 Effect of 16H7 on Body Mass Index (BMI), AbdominalCircumference (AC) and Skin Fold Thickness (SFT)

BMI, AC and SFT were monitored weekly throughout the study, both pre-and post-administration of test compound when the body weight was taken.BMI is defined as the individual's body weight divided by the square ofhis or her height. SFT is the thickness of a double layer of skin andthe fat beneath it as measured with a caliper. BMI, SFT and AC arerelatively accurate, simple, and inexpensive measurements of bodycomposition, particularly indicative of subcutaneous fat. Animalstreated with vehicle showed relatively stable BMI, SFT and AC throughoutthe study. Animals treated with 16H7 showed decreased levels of BMI, ACand SFT over the course of the 4 week study, suggesting that 16H7compound resulted in reduction of fat mass. Results are shown in FIGS.18-20, respectively. These measured parameters did not come back tobaseline values at the end of the 6 weeks wash out period.

Example 13.5 Effect of 16H7 on Oral Glucose Tolerance Test (OGTT)

OGTTs were conducted before and after initiation of treatments. Before16H7 injections baseline values for glucose and insulin levels weremeasured throughout the OGTT (FIGS. 21 and 22, respectively) and werenot statistically significantly different between the vehicle and 16H7groups. Post-dose OGTTs were performed every two weeks during thetreatment period and after 3 weeks of wash out period. 16H7 slightlyimproved glucose tolerance after 4 weeks of treatment and 3 weeks ofwash out period. The animal model used is not glucose intolerantexplaining the modest effects observed (FIG. 21). Insulin levels werestatistically significantly decreased in animals treated with 16H7(significance observed at time 0 during the OGTT performed after 2 weeksof treatment, at time 0 and 15 minutes during the OGTT performed after 4weeks of treatment and at time 0 and 60 minutes during the OGTTperformed after 2 weeks of treatment) (FIG. 22).

Example 13.6 Effect of 16H7 on Fasting and Fed Blood Glucose and InsulinLevels

Blood was collected from overnight fasted animals or in fed conditionsafter the AM feeding. In the fasted conditions, blood drawn wasconducted weekly 5 days post each injection. In the fed conditions,blood drawn was conducted on days 2, 11, 16, 25 and 46 post firstinjection. 16H7 did not reduce fasting or fed blood glucose levels(FIGS. 23 and 25). No hypoglycemia was observed in any of the monkeystreated with 16H7. 16H7 did, however, result in a statisticallysignificant decrease in fasting and fed plasma insulin levels (FIGS. 24and 26).

Example 13.7 Effect of 16H7 on Triglyceride Levels

Measurements were made from the same samples collected for glucose andinsulin measurements. Triglyceride levels were significantly reduced inanimals treated with 16H7 when measured in fasted or fed conditions(FIGS. 27 and 28).

Example 13.8 Conclusions

In a study conducted in male obese cynomolgus monkeys, animals treatedwith 16H7 showed improved metabolic parameters. Body weight was reducedand body composition was improved. Short-term reduction of food intakewas observed and the effect diminished and the food intake recovered tobaseline or control levels at 21 days into the study. Fasting insulinand triglyceride levels were also reduced by 16H7. Insulin levelsmeasured during OGTT were also improved.

Example 14 Variant Forms of Antigen Binding Proteins 16H7 and 22H5

Antigen binding proteins 16H7 and 22H5, which are described herein inTables 1-4, were mutated to impart different properties to the molecule,such as changes in solubility, pI, overall charge, immunogenicity inhumans and in animal models, stability, etc. The mutations comprisedadditions, deletions or substitutions in either the light chain(designated “LC”, SEQ ID NO:14) or heavy chain (designated “HC”, SEQ IDNO:32) of the molecule. The disclosed single point mutations were madeindividually or two or more mutations were combined.

Examples of mutations and combinations of mutations that were introducedinto the 16H7 heavy and light chain sequences include the following:

-   -   I83K (in 16H7 heavy chain) (SEQ ID NO:396)    -   E16Q (in 16H7 heavy chain)+V24F (in 16H7 heavy chain)+I83T (in        16H7 heavy chain)+S100 I (in 16H7 heavy chain)+T119L (in 16H7        heavy chain) (SEQ ID NO:395)    -   D109S (in 16H7 heavy chain) (SEQ ID NO:401)    -   Deletion of Y107 (in 16H7 heavy chain) (SEQ ID NO:400)    -   Insertion of a Y residue on the N-terminal side of Y107 (in 16H7        heavy chain) (SEQ ID NO:405)    -   D88R+P89A+V90E (in 16H7 heavy chain) (SEQ ID NO:398)    -   D49Y (in 16H7 light chain) (SEQ ID NO:386)    -   D49A (in 16H7 light chain) (SEQ ID NO:387)    -   D91A (in 16H7 light chain) (SEQ ID NO:388)    -   D49A (in 16H7 light chain)+D91A (in 16H7 light chain) (SEQ ID        NO:389)    -   Q16K (in 16H7 light chain) (SEQ ID NO:385)

Examples of mutations and combinations of mutations that were introducedinto the 22H5 heavy and light chain sequences include the following:

N92Q (in 22H5 light chain) (SEQ ID NO:402)

S94A (in 22H5 light chain) (SEQ ID NO:403)

C109S (in 22H5 heavy chain) (SEQ ID NO:404)

Summarily, the generated antigen binding proteins comprised thefollowing pairs of 16H7 heavy and light chains:

-   -   (i) 16H7 light chain (SEQ ID NO:14) paired with a 16H7 heavy        chain comprising I83K (SEQ ID NO:396);    -   (ii) 16H7 light chain (SEQ ID NO:14) paired with a 16H7 heavy        chain comprising E16Q, V24F, I83T, S100I, T119L (SEQ ID NO:395);    -   (iii) 16H7 light chain (SEQ ID NO:14) paired with a 16H7 heavy        chain comprising D109S (SEQ ID NO:401);    -   (iv) 16H7 light chain (SEQ ID NO:14) paired with a 16H7 heavy        chain comprising the deletion of Y107 (SEQ ID NO:400);    -   (v) 16H7 light chain (SEQ ID NO:14) paired with a 16H7 heavy        chain comprising the insertion of a Y residue on the N-terminal        side of Y107 (SEQ ID NO:405);    -   (vi) 16H7 light chain (SEQ ID NO:14) paired with a 16H7 heavy        chain comprising D88R, P89A, V90E, (SEQ ID NO:398);    -   (vii) 16H7 heavy chain (SEQ ID NO:32) paired with a 16H7 light        chain comprising D49Y (SEQ ID NO:386);    -   (viii) 16H7 heavy chain (SEQ ID NO:32) paired with a 16H7 light        chain comprising D49A (LC) (SEQ ID NO:387);    -   (xi) 16H7 heavy chain (SEQ ID NO:32) paired with a 16H7 light        chain comprising D91A (SEQ ID NO:388);    -   (ix) 16H7 heavy chain (SEQ ID NO:32) paired with a 16H7 light        chain comprising D49A, D91A (SEQ ID NO:389);    -   (x) 16H7 heavy chain (SEQ ID NO:32) paired with a 16H7 light        chain comprising Q16K (LC) (SEQ ID NO:385);    -   and the following pairs of 22H5 heavy and light chain sequences:    -   (xi) 22H5 heavy chain (SEQ ID NO:31) paired with a 22H5 light        chain comprising N92Q (LC) (SEQ ID NO:402);    -   (xii) 22H5 heavy chain (SEQ ID NO:31) paired with a 22H5 light        chain comprising S94A (LC) (SEQ ID NO:403);    -   (xiii) 22H5 light chain (SEQ ID NO:13) paired with a 22H5 heavy        chain comprising C109S(HC) (SEQ ID NO:404);    -   (xiv) 22H5 light chain (SEQ ID NO:13) paired with a 22H5 heavy        chain comprising an insertion of a tyrosine residue at position        107 (SEQ ID NO:405).

The amino acid sequences for the generated light chain variants areshown in Table 6:

TABLE 6A Amino Acid Sequences of 16H7 and 22H5 Variants SEQ ID NO of SEQCore Paired Paired Amino Acid Sequence of Variant ID Sequence VariationWith Sequence Chain NO: 16H7 Q16K H3 32 SYVLTQPPSVSVAPG K TARITCGGN 385light NIGSESVHWYQQKPGQAPVLVVYD chain DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGNSDHVV FGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS 16H7 D49Y H3 32SYVLTQPPSVSVAPGQTARITCGGN 386 light NIGSESVHWYQQKPGQAPVLVVY Y chainDSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGNSDHVVFGGGTKLTVLGQPKANPTVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSN NKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS16H7 D49A H3 32 SYVLTQPPSVSVAPGQTARITCGGN 387 lightNIGSESVHWYQQKPGQAPVLVVY A chain DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGNSDHVV FGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS 16H7 Q16K H3 32SYVLTQPPSVSVAPG K TARITCGGN 385 light NIGSESVHWYQQKPGQAPVLVVYD chainDSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGNSDHVVFGGGTKLTVLGQPKANPTVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSN NKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS16H7 D49Y H3 32 SYVLTQPPSVSVAPGQTARITCGGN 386 lightNIGSESVHWYQQKPGQAPVLVVY Y chain DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGNSDHVV FGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS 16H7 D91A H3 32SYVLTQPPSVSVAPGQTARITCGGN 388 light NIGSESVHWYQQKPGQAPVLVVYD chainDSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVW A GNSDHVVFGGGTKLTVLGQPKANPTVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSN NKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS16H7 Q16K H3 32 SYVLTQPPSVSVAPG K TARITCGGN 385 lightNIGSESVHWYQQKPGQAPVLVVYD chain DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGNSDHVV FGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS 16H7 D49Y H3 32SYVLTQPPSVSVAPGQTARITCGGN 386 light NIGSESVHWYQQKPGQAPVLVVY Y chainDSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGNSDHVVFGGGTKLTVLGQPKANPTVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSN NKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS16H7 D49A + H3 32 SYVLTQPPSVSVAPGQTARITCGGN 389 light D91ANIGSESVHWYQQKPGQAPVLVVY A chain DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVW A GNSDHVV FGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS 16H7 Q16K H3 32SYVLTQPPSVSVAPG K TARITCGGN 385 light NIGSESVHWYQQKPGQAPVLVVYD chainDSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGNSDHVVFGGGTKLTVLGQPKANPTVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSN NKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS16H7 D49Y H3 32 SYVLTQPPSVSVAPGQTARITCGGN 386 lightNIGSESVHWYQQKPGQAPVLVVY Y chain DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGNSDHVV FGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS 16H7 V24F L3 14QVTLKESGPVLVKPTETLTLTCT F S 390 heavy GFSLNNARMGVSWIRQPPGKALEW chainLAHIFSNDEKSYSTSLKSRLTISKDT SKSQVVLIMTNMDPVDTATYYCARSVVTGGYYYDGMDVWGQGTTVT VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDK TVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWL NGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 16H7 Q16KH3 32 SYVLTQPPSVSVAPG K TARITCGGN 385 light NIGSESVHWYQQKPGQAPVLVVYDchain DSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGNSDHVVFGGGTKLTVLGQPKANPTVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSN NKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS16H7 D49Y H3 32 SYVLTQPPSVSVAPGQTARITCGGN 386 lightNIGSESVHWYQQKPGQAPVLVVY Y chain DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGNSDHVV FGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS 16H7 I83T L3 14QVTLKESGPVLVKPTETLTLTCTVS 391 heavy GFSLNNARMGVSWIRQPPGKALEW chainLAHIFSNDEKSYSTSLKSRLTISKDT SKSQVVL T MTNMDPVDTATYYCARSVVTGGYYYDGMDVWGQGTTVT VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDK TVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWL NGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 16H7 Q16KH3 32 SYVLTQPPSVSVAPG K TARITCGGN 385 light NIGSESVHWYQQKPGQAPVLVVYDchain DSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGNSDHVVFGGGTKLTVLGQPKANPTVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSN NKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS16H7 D49Y H3 32 SYVLTQPPSVSVAPGQTARITCGGN 386 lightNIGSESVHWYQQKPGQAPVLVVY Y chain DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGNSDHVV FGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS 16H7 V24F + L3 14QVTLKESGPVLVKPTETLTLTCT F S 392 heavy I83T GFSLNNARMGVSWIRQPPGKALEWchain LAHIFSNDEKSYSTSLKSRLTISKDT SKSQVVL T MTNMDPVDTATYYCARSVVTGGYYYDGMDVWGQGTTVT VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDK TVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWL NGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 16H7 Q16KH3 32 SYVLTQPPSVSVAPG K TARITCGGN 385 light NIGSESVHWYQQKPGQAPVLVVYDchain DSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGNSDHVVFGGGTKLTVLGQPKANPTVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSN NKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS16H7 D49Y H3 32 SYVLTQPPSVSVAPGQTARITCGGN 386 lightNIGSESVHWYQQKPGQAPVLVVY Y chain DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGNSDHVV FGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS 16H7 E16Q + L3 14QVTLKESGPVLVKPT Q TLTLTCT F S 393 heavy V24F + GFSLNNARMGVSWIRQPPGKALEWchain I83T LAHIFSNDEKSYSTSLKSRLTISKDT SKSQVVL T MTNMDPVDTATYYCARSVVTGGYYYDGMDVWGQGTTVT VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDK TVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWL NGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 16H7 Q16KH3 32 SYVLTQPPSVSVAPG K TARITCGGN 385 light NIGSESVHWYQQKPGQAPVLVVYDchain DSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGNSDHVVFGGGTKLTVLGQPKANPTVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSN NKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS16H7 D49Y H3 32 SYVLTQPPSVSVAPGQTARITCGGN 386 lightNIGSESVHWYQQKPGQAPVLVVY Y chain DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGNSDHVV FGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS 16H7 E16Q + L3 14QVTLKESGPVLVKPT Q TLTLTCT F S 394 heavy V24F + GFSLNNARMGVSWIRQPPGKALEWchain I83T + LAHIFSNDEKSYSTSLKSRLTISKDT T119L SKSQVVL T MTNMDPVDTATYYCARSVVTGGYYYDGMDVWGQGT L V TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVD KTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW LNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 16H7 Q16KH3 32 SYVLTQPPSVSVAPG K TARITCGGN 385 light NIGSESVHWYQQKPGQAPVLVVYDchain DSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGNSDHVVFGGGTKLTVLGQPKANPTVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSN NKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS16H7 D49Y H3 32 SYVLTQPPSVSVAPGQTARITCGGN 386 lightNIGSESVHWYQQKPGQAPVLVVY Y chain DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGNSDHVV FGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS 16H7 E16Q + L3 14QVTLKESGPVLVKPT Q TLTLTCT F S 395 heavy V24F + GFSLNNARMGVSWIRQPPGKALEWchain I83T + LAHIFSNDEKSYSTSLKSRLTISKDT S100I + SKSQVVL TMTNMDPVDTATYYCA T119L R I VVTGGYYYDGMDVWGQGT L VTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKT KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 16H7 Q16K H3 32 SYVLTQPPSVSVAPG KTARITCGGN 385 light NIGSESVHWYQQKPGQAPVLVVYD chainDSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGNSDHVVFGGGTKLTVLGQPKANPTVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSN NKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS16H7 D49Y H3 32 SYVLTQPPSVSVAPGQTARITCGGN 386 lightNIGSESVHWYQQKPGQAPVLVVY Y chain DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGNSDHVV FGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS 16H7 I83K L3 14QVTLKESGPVLVKPTETLTLTCTVS 396 heavy GFSLNNARMGVSWIRQPPGKALEW chainLAHIFSNDEKSYSTSLKSRLTISKDT SKSQVVL K MTNMDPVDTATYYCARSVVTGGYYYDGMDVWGQGTTVT VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDK TVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWL NGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 16H7 Q16KH3 32 SYVLTQPPSVSVAPG K TARITCGGN 385 light NIGSESVHWYQQKPGQAPVLVVYDchain DSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGNSDHVVFGGGTKLTVLGQPKANPTVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSN NKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS16H7 D49Y H3 32 SYVLTQPPSVSVAPGQTARITCGGN 386 lightNIGSESVHWYQQKPGQAPVLVVY Y chain DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGNSDHVV FGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS 16H7 S100I L3 14QVTLKESGPVLVKPTETLTLTCTVS 397 heavy GFSLNNARMGVSWIRQPPGKALEW chainLAHIFSNDEKSYSTSLKSRLTISKDT SKSQVVLIMTNMDPVDTATYYCAR IVVTGGYYYDGMDVWGQGTTVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKT VERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLN GKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 16H7 Q16KH3 32 SYVLTQPPSVSVAPG K TARITCGGN 385 light NIGSESVHWYQQKPGQAPVLVVYDchain DSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGNSDHVVFGGGTKLTVLGQPKANPTVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSN NKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS16H7 D49Y H3 32 SYVLTQPPSVSVAPGQTARITCGGN 386 lightNIGSESVHWYQQKPGQAPVLVVY Y chain DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGNSDHVV FGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS 16H7 D88R + L3 14QVTLKESGPVLVKPTETLTLTCTVS 398 heavy P89A + GFSLNNARMGVSWIRQPPGKALEWchain V90E LAHIFSNDEKSYSTSLKSRLTISKDT SKSQVVLIMTNM RAE DTATYYCARSVVTGGYYYDGMDVWGQGTTVT VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDK TVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWL NGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 16H7 Q16KH3 32 SYVLTQPPSVSVAPG K TARITCGGN 385 light NIGSESVHWYQQKPGQAPVLVVYDchain DSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGNSDHVVFGGGTKLTVLGQPKANPTVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSN NKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS16H7 D49Y H3 32 SYVLTQPPSVSVAPGQTARITCGGN 386 lightNIGSESVHWYQQKPGQAPVLVVY Y chain DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGNSDHVV FGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS 16H7 D88R + L3 14QVTLKESGPVLVKPTETLTLTCTVS 399 heavy P89A + GFSLNNARMGVSWIRQPPGKALEWchain V90E + LAHIFSNDEKSYSTSLKSRLTISKDT S100I SKSQVVLIMTNM RAE DTATYYCARI VVTGGYYYDGMDVWGQGTTVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKT VERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLN GKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 16H7 Q16KH3 32 SYVLTQPPSVSVAPG K TARITCGGN 385 light NIGSESVHWYQQKPGQAPVLVVYDchain DSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGNSDHVVFGGGTKLTVLGQPKANPTVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSN NKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS16H7 D49Y H3 32 SYVLTQPPSVSVAPGQTARITCGGN 386 lightNIGSESVHWYQQKPGQAPVLVVY Y chain DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGNSDHVV FGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS 16H7 Deletion L3 14QVTLKESGPVLVKPTETLTLTCTVS 400 heavy of GFSLNNARMGVSWIRQPPGKALEW chainY107 LAHIFSNDEKSYSTSLKSRLTISKDT SKSQVVLIMTNMDPVDTATYYCARSVVTGGYYDGMDVWGQGTTVTVS SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNG KEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK 16H7 Q16K H332 SYVLTQPPSVSVAPG K TARITCGGN 385 light NIGSESVHWYQQKPGQAPVLVVYD chainDSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGNSDHVVFGGGTKLTVLGQPKANPTVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSN NKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS16H7 D49Y H3 32 SYVLTQPPSVSVAPGQTARITCGGN 386 lightNIGSESVHWYQQKPGQAPVLVVY Y chain DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGNSDHVV FGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS 16H7 D109S L3 14QVTLKESGPVLVKPTETLTLTCTVS 401 heavy GFSLNNARMGVSWIRQPPGKALEW chainLAHIFSNDEKSYSTSLKSRLTISKDT SKSQVVLIMTNMDPVDTATYYCAR SVVTGGYYY SGMDVWGQGTTVTV SSASTKGPSVFPLAPCSRSTSESTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 16H7 Q16K H3 32 SYVLTQPPSVSVAPG KTARITCGGN 385 light NIGSESVHWYQQKPGQAPVLVVYD chainDSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGNSDHVVFGGGTKLTVLGQPKANPTVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSN NKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS16H7 D49Y H3 32 SYVLTQPPSVSVAPGQTARITCGGN 386 lightNIGSESVHWYQQKPGQAPVLVVY Y chain DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGNSDHVV FGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS 22H5 N92Q H2 31SYVLTQPPSVSVAPGQTARITCGGN 402 light NIGSQSVHWYQQKPGQAPVLVVY chainDDSDRPSGIPERFSGSNSGNTATLTI SRVEAGDEADYYCQVWD Q TSDHVVFGGGTKLTVLGQPKANPTVTLFPP SSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQS NNKYAASSYLSLTPEQWKSHRSYS CQVTHEGSTVEKTVAPTECS16H7 Q16K H3 32 SYVLTQPPSVSVAPG K TARITCGGN 385 lightNIGSESVHWYQQKPGQAPVLVVYD chain DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGNSDHVV FGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS 16H7 D49Y H3 32SYVLTQPPSVSVAPGQTARITCGGN 386 light NIGSESVHWYQQKPGQAPVLVVY Y chainDSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGNSDHVVFGGGTKLTVLGQPKANPTVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSN NKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS22H5 S94A H2 31 SYVLTQPPSVSVAPGQTARITCGGN 403 lightNIGSQSVHWYQQKPGQAPVLVVY chain DDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDNT A DHV VFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVT VAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYS CQVTHEGSTVEKTVAPTECS 16H7 Q16K H3 32SYVLTQPPSVSVAPG K TARITCGGN 385 light NIGSESVHWYQQKPGQAPVLVVYD chainDSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGNSDHVVFGGGTKLTVLGQPKANPTVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSN NKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS16H7 D49Y H3 32 SYVLTQPPSVSVAPGQTARITCGGN 386 lightNIGSESVHWYQQKPGQAPVLVVY Y chain DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGNSDHVV FGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS 22H5 C109S L2 13QVTLKESGPVLVKPTETLTLTCTVS 404 heavy GFSLSNARMGVSWIRQPPGKALEW chainLAHIFSNDEKSYSTSLKSRLTISKDT SKSQVVLTMTNMDPVDTATYYCA RILLVGAYYY SGMDVWGQGTTVT VSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKT KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 16H7 Q16K H3 32 SYVLTQPPSVSVAPG KTARITCGGN 385 light NIGSESVHWYQQKPGQAPVLVVYD chainDSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGNSDHVVFGGGTKLTVLGQPKANPTVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSN NKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS16H7 D49Y H3 32 SYVLTQPPSVSVAPGQTARITCGGN 386 lightNIGSESVHWYQQKPGQAPVLVVY Y chain DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGNSDHVV FGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS 16H7 Insertion L3 14QVTLKESGPVLVKPTETLTLTCTVS 405 heavy of GFSLNNARMGVSWIRQPPGKALEW chainY107 LAHIFSNDEKSYSTSLKSRLTISKDT SKSQVVLIMTNMDPVDTATYYCARSVVTGGYYYYDGMDVWGQGTTV TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVD KTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW LNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK

TABLE 6B Nucleic Acid Sequences of 16H7 and 22H5 Variants Core SequenceVariation Nucleic Acid Sequence SEQ ID NO: 16H7 Q16KTCCTATGTGCTGACTCAGCCACCCTCGGTGT 406 light CAGTGGCCCCAGGA AAGACGGCCAGGATT chain ACCTGTGGGGGAAACAACATTGGAAGTGAAAGTGTGCACTGGTACCAGCAGAAGCCAGG CCAGGCCCCTGTGCTGGTCGTCTATGATGATAGCGACCGGCCCTCAGGGATCCCTGAGCG ATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGG GGATGAGGCCGACTATTACTGTCAGGTGTGGGATGGTAATAGTGACCATGTGGTATTCGG CGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCCAACCCCACTGTCACTCTGTT CCCGCCCTCCTCTGAGGAGCTCCAAGCCAACAAGGCCACACTAGTGTGTCTGATCAGTGA CTTCTACCCGGGAGCTGTGACAGTGGCCTGGAAGGCAGATGGCAGCCCCGTCAAGGCGG GAGTGGAGACCACCAAACCCTCCAAACAGAGCAACAACAAGTACGCGGCCAGCAGCTA CCTGAGCCTGACGCCCGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCA TGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA 16H7 D49Y TCCTATGTGCTGACTCAGCCACCCTCGGTGT 407 lightCAGTGGCCCCAGGACAGACGGCCAGGATTA chain CCTGTGGGGGAAACAACATTGGAAGTGAAAGTGTGCACTGGTACCAGCAGAAGCCAGGC CAGGCCCCTGTGCTGGTCGTCTAT TAT GATAGCGACCGGCCCTCAGGGATCCCTGAGCGA TTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGG GATGAGGCCGACTATTACTGTCAGGTGTGGGATGGTAATAGTGACCATGTGGTATTCGGC GGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCCAACCCCACTGTCACTCTGTTC CCGCCCTCCTCTGAGGAGCTCCAAGCCAACAAGGCCACACTAGTGTGTCTGATCAGTGAC TTCTACCCGGGAGCTGTGACAGTGGCCTGGAAGGCAGATGGCAGCCCCGTCAAGGCGGG AGTGGAGACCACCAAACCCTCCAAACAGAGCAACAACAAGTACGCGGCCAGCAGCTACC TGAGCCTGACGCCCGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATG AAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA 16H7 D49A TCCTATGTGCTGACTCAGCCACCCTCGGTGT 408 lightCAGTGGCCCCAGGACAGACGGCCAGGATTA chain CCTGTGGGGGAAACAACATTGGAAGTGAAAGTGTGCACTGGTACCAGCAGAAGCCAGGC CAGGCCCCTGTGCTGGTCGTCTAT GCT GATAGCGACCGGCCCTCAGGGATCCCTGAGCGA TTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGG GATGAGGCCGACTATTACTGTCAGGTGTGGGATGGTAATAGTGACCATGTGGTATTCGGC GGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCCAACCCCACTGTCACTCTGTTC CCGCCCTCCTCTGAGGAGCTCCAAGCCAACAAGGCCACACTAGTGTGTCTGATCAGTGAC TTCTACCCGGGAGCTGTGACAGTGGCCTGGAAGGCAGATGGCAGCCCCGTCAAGGCGGG AGTGGAGACCACCAAACCCTCCAAACAGAGCAACAACAAGTACGCGGCCAGCAGCTACC TGAGCCTGACGCCCGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATG AAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA 16H7 D91A TCCTATGTGCTGACTCAGCCACCCTCGGTGT 409 lightCAGTGGCCCCAGGACAGACGGCCAGGATTA chain CCTGTGGGGGAAACAACATTGGAAGTGAAAGTGTGCACTGGTACCAGCAGAAGCCAGGC CAGGCCCCTGTGCTGGTCGTCTATGATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGA TTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGG GATGAGGCCGACTATTACTGTCAGGTGTGG GCTGGTAATAGTGACCATGTGGTATTCGGC GGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCCAACCCCACTGTCACTCTGTTC CCGCCCTCCTCTGAGGAGCTCCAAGCCAACAAGGCCACACTAGTGTGTCTGATCAGTGAC TTCTACCCGGGAGCTGTGACAGTGGCCTGGAAGGCAGATGGCAGCCCCGTCAAGGCGGG AGTGGAGACCACCAAACCCTCCAAACAGAGCAACAACAAGTACGCGGCCAGCAGCTACC TGAGCCTGACGCCCGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATG AAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA 16H7 D49A + D91A TCCTATGTGCTGACTCAGCCACCCTCGGTGT 410light CAGTGGCCCCAGGACAGACGGCCAGGATTA chain CCTGTGGGGGAAACAACATTGGAAGTGAAAGTGTGCACTGGTACCAGCAGAAGCCAGGC CAGGCCCCTGTGCTGGTCGTCTAT GCT GATAGCGACCGGCCCTCAGGGATCCCTGAGCGA TTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGG GATGAGGCCGACTATTACTGTCAGGTGTGG GCTGGTAATAGTGACCATGTGGTATTCGGC GGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCCAACCCCACTGTCACTCTGTTC CCGCCCTCCTCTGAGGAGCTCCAAGCCAACAAGGCCACACTAGTGTGTCTGATCAGTGAC TTCTACCCGGGAGCTGTGACAGTGGCCTGGAAGGCAGATGGCAGCCCCGTCAAGGCGGG AGTGGAGACCACCAAACCCTCCAAACAGAGCAACAACAAGTACGCGGCCAGCAGCTACC TGAGCCTGACGCCCGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATG AAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA 16H7 V24F CAGGTCACCTTGAAGGAGTCTGGTCCTGTG 411 heavyCTGGTGAAACCCACAGAGACCCTCACGCTG chain ACCTGCACCTTCTCTGGGTTCTCACTCAACAATGCTAGAATGGGTGTGAGCTGGATCCGTC AGCCCCCAGGGAAGGCCCTGGAGTGGCTTGCACACATTTTTTCGAATGACGAAAAATCCT ACAGCACATCTCTGAAGAGCAGGCTCACCATCTCCAAGGACACCTCCAAAAGCCAGGTGG TCCTAATTATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACGGTCAG TAGTAACTGGCGGCTACTACTACGACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCA CCGTCTCTAGTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGA GCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCC TACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCG GCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAG ACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGA CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGT ACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAAC AGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAG GAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGA GATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACAT CGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCA TGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGT GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA CGCAGAAGAGCCTCTCCCTGTCTCCGGGTA AA 16H7I83T CAGGTCACCTTGAAGGAGTCTGGTCCTGTG 412 heavyCTGGTGAAACCCACAGAGACCCTCACGCTG chain ACCTGCACCGTGTCTGGGTTCTCACTCAACAATGCTAGAATGGGTGTGAGCTGGATCCGT CAGCCCCCAGGGAAGGCCCTGGAGTGGCTTGCACACATTTTTTCGAATGACGAAAAATCC TACAGCACATCTCTGAAGAGCAGGCTCACCATCTCCAAGGACACCTCCAAAAGCCAGGTG GTCCTAACCATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACGGTCA GTAGTAACTGGCGGCTACTACTACGACGGTATGGACGTCTGGGGCCAAGGGACCACGGTC ACCGTCTCTAGTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGA GCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCC TACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCG GCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAG ACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGA CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGT ACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAAC AGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAG GAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGA GATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACAT CGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCA TGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGT GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA CGCAGAAGAGCCTCTCCCTGTCTCCGGGTA AA 16H7V24F + I83T CAGGTCACCTTGAAGGAGTCTGGTCCTGTG 413 heavyCTGGTGAAACCCACAGAGACCCTCACGCTG chain ACCTGCACCTTCTCTGGGTTCTCACTCAACAATGCTAGAATGGGTGTGAGCTGGATCCGTC AGCCCCCAGGGAAGGCCCTGGAGTGGCTTGCACACATTTTTTCGAATGACGAAAAATCCT ACAGCACATCTCTGAAGAGCAGGCTCACCATCTCCAAGGACACCTCCAAAAGCCAGGTGG TCCTAACCATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACGGTCAG TAGTAACTGGCGGCTACTACTACGACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCA CCGTCTCTAGTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGA GCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCC TACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCG GCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAG ACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGA CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGT ACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAAC AGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAG GAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGA GATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACAT CGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCA TGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGT GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA CGCAGAAGAGCCTCTCCCTGTCTCCGGGTA AA 16H7E16Q + V24F + CAGGTCACCTTGAAGGAGTCTGGTCCTGTG 414 heavy I83TCTGGTGAAACCCACACAGACCCTCACGCTG chain ACCTGCACCTTCTCTGGGTTCTCACTCAACAATGCTAGAATGGGTGTGAGCTGGATCCGTC AGCCCCCAGGGAAGGCCCTGGAGTGGCTTGCACACATTTTTTCGAATGACGAAAAATCCT ACAGCACATCTCTGAAGAGCAGGCTCACCATCTCCAAGGACACCTCCAAAAGCCAGGTGG TCCTAACCATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACGGTCAG TAGTAACTGGCGGCTACTACTACGACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCA CCGTCTCTAGTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGA GCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCC TACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCG GCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAG ACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGA CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGT ACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAAC AGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAG GAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGA GATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACAT CGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCA TGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGT GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA CGCAGAAGAGCCTCTCCCTGTCTCCGGGTA AA 16H7E16Q + V24F + CAGGTCACCTTGAAGGAGTCTGGTCCTGTG 415 heavy I83T + T119LCTGGTGAAACCCACACAGACCCTCACGCTG chain ACCTGCACCTTCTCTGGGTTCTCACTCAACAATGCTAGAATGGGTGTGAGCTGGATCCGTC AGCCCCCAGGGAAGGCCCTGGAGTGGCTTGCACACATTTTTTCGAATGACGAAAAATCCT ACAGCACATCTCTGAAGAGCAGGCTCACCATCTCCAAGGACACCTCCAAAAGCCAGGTGG TCCTAACCATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACGGTCAG TAGTAACTGGCGGCTACTACTACGACGGTATGGACGTCTGGGGCCAAGGGACCCTGGTCA CCGTCTCTAGTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGA GCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCC TACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCG GCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAG ACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGA CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGT ACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAAC AGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAG GAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGA GATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACAT CGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCA TGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGT GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA CGCAGAAGAGCCTCTCCCTGTCTCCGGGTA AA 16H7E16Q + V24F + CAGGTCACCTTGAAGGAGTCTGGTCCTGTG 416 heavy I83T + S100I +CTGGTGAAACCCACACAGACCCTCACGCTG chain T119LACCTGCACCTTCTCTGGGTTCTCACTCAACA ATGCTAGAATGGGTGTGAGCTGGATCCGTCAGCCCCCAGGGAAGGCCCTGGAGTGGCTTG CACACATTTTTTCGAATGACGAAAAATCCTACAGCACATCTCTGAAGAGCAGGCTCACCA TCTCCAAGGACACCTCCAAAAGCCAGGTGGTCCTAACCATGACCAACATGGACCCTGTGG ACACAGCCACATATTACTGTGCACGGATCGTAGTAACTGGCGGCTACTACTACGACGGTA TGGACGTCTGGGGCCAAGGGACCCTGGTCACCGTCTCTAGTGCCTCCACCAAGGGCCCAT CGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCT GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGA CCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCA GCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATC ACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGC CCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCC ACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCA AGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACC GTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGC CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACA GGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTG CCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC CGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTA CAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCG TGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTA AA 16H7 I83KCAGGTCACCTTGAAGGAGTCTGGTCCTGTG 417 heavy CTGGTGAAACCCACAGAGACCCTCACGCTGchain ACCTGCACCGTGTCTGGGTTCTCACTCAAC AATGCTAGAATGGGTGTGAGCTGGATCCGTCAGCCCCCAGGGAAGGCCCTGGAGTGGCTT GCACACATTTTTTCGAATGACGAAAAATCCTACAGCACATCTCTGAAGAGCAGGCTCACC ATCTCCAAGGACACCTCCAAAAGCCAGGTGGTCCTAAAGATGACCAACATGGACCCTGTG GACACAGCCACATATTACTGTGCACGGTCAGTAGTAACTGGCGGCTACTACTACGACGGT ATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCTAGTGCCTCCACCAAGGGCCCA TCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCT GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGA CCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCA GCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATC ACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGC CCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCC ACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCA AGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACC GTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGC CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACA GGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTG CCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC CGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTA CAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCG TGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTA AA 16H7 S100ICAGGTCACCTTGAAGGAGTCTGGTCCTGTG 418 heavy CTGGTGAAACCCACAGAGACCCTCACGCTGchain ACCTGCACCGTGTCTGGGTTCTCACTCAAC AATGCTAGAATGGGTGTGAGCTGGATCCGTCAGCCCCCAGGGAAGGCCCTGGAGTGGCTT GCACACATTTTTTCGAATGACGAAAAATCCTACAGCACATCTCTGAAGAGCAGGCTCACC ATCTCCAAGGACACCTCCAAAAGCCAGGTGGTCCTAATTATGACCAACATGGACCCTGTG GACACAGCCACATATTACTGTGCACGGATCGTAGTAACTGGCGGCTACTACTACGACGGT ATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCTAGTGCCTCCACCAAGGGCCCA TCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCT GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGA CCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCA GCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATC ACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGC CCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCC ACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCA AGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACC GTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGC CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACA GGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTG CCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC CGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTA CAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCG TGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTA AA 16H7 D88R + P89A +CAGGTCACCTTGAAGGAGTCTGGTCCTGTG 419 heavy V90ECTGGTGAAACCCACAGAGACCCTCACGCTG chain ACCTGCACCGTGTCTGGGTTCTCACTCAACAATGCTAGAATGGGTGTGAGCTGGATCCGT CAGCCCCCAGGGAAGGCCCTGGAGTGGCTTGCACACATTTTTTCGAATGACGAAAAATCC TACAGCACATCTCTGAAGAGCAGGCTCACCATCTCCAAGGACACCTCCAAAAGCCAGGTG GTCCTAATTATGACCAACATGAGAGCTGAGGACACAGCCACATATTACTGTGCACGGTCA GTAGTAACTGGCGGCTACTACTACGACGGTATGGACGTCTGGGGCCAAGGGACCACGGTC ACCGTCTCTAGTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGA GCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCC TACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCG GCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAG ACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGA CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGT ACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAAC AGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAG GAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGA GATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACAT CGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCA TGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGT GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA CGCAGAAGAGCCTCTCCCTGTCTCCGGGTA AA 16H7D88R + P89A + CAGGTCACCTTGAAGGAGTCTGGTCCTGTG 420 heavy V90E + S100ICTGGTGAAACCCACAGAGACCCTCACGCTG chain ACCTGCACCGTGTCTGGGTTCTCACTCAACAATGCTAGAATGGGTGTGAGCTGGATCCGT CAGCCCCCAGGGAAGGCCCTGGAGTGGCTTGCACACATTTTTTCGAATGACGAAAAATCC TACAGCACATCTCTGAAGAGCAGGCTCACCATCTCCAAGGACACCTCCAAAAGCCAGGTG GTCCTAATTATGACCAACATGAGAGCTGAGGACACAGCCACATATTACTGTGCACGGATC GTAGTAACTGGCGGCTACTACTACGACGGTATGGACGTCTGGGGCCAAGGGACCACGGTC ACCGTCTCTAGTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGA GCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCC TACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCG GCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAG ACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGA CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGT ACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAAC AGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAG GAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGA GATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACAT CGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCA TGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGT GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA CGCAGAAGAGCCTCTCCCTGTCTCCGGGTA AA 16H7Deletion of CAGGTCACCTTGAAGGAGTCTGGTCCTGTG 421 heavy Y107CTGGTGAAACCCACAGAGACCCTCACGCTG chain ACCTGCACCGTGTCTGGGTTCTCACTCAACAATGCTAGAATGGGTGTGAGCTGGATCCGT CAGCCCCCAGGGAAGGCCCTGGAGTGGCTTGCACACATTTTTTCGAATGACGAAAAATCC TACAGCACATCTCTGAAGAGCAGGCTCACCATCTCCAAGGACACCTCCAAAAGCCAGGTG GTCCTAATTATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACGGTCA GTAGTAACTGGCGGCTAC|TACGACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACC GTCTCTAGTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGC ACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG ACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTA CAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGC ACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAC AGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACC GTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAG GTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTAC GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAG CACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGA GTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAA AACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGA TGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCG CCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATG CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGG CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACG CAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 16H7 D109SCAGGTCACCTTGAAGGAGTCTGGTCCTGTG 422 heavy CTGGTGAAACCCACAGAGACCCTCACGCTGchain ACCTGCACCGTGTCTGGGTTCTCACTCAAC AATGCTAGAATGGGTGTGAGCTGGATCCGTCAGCCCCCAGGGAAGGCCCTGGAGTGGCTT GCACACATTTTTTCGAATGACGAAAAATCCTACAGCACATCTCTGAAGAGCAGGCTCACC ATCTCCAAGGACACCTCCAAAAGCCAGGTGGTCCTAATTATGACCAACATGGACCCTGTG GACACAGCCACATATTACTGTGCACGGTCAGTAGTAACTGGCGGCTACTACTACAGCGGT ATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCTAGTGCCTCCACCAAGGGCCCA TCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCT GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGA CCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCA GCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATC ACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGC CCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCC ACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCA AGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACC GTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGC CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACA GGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTG CCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC CGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTA CAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCG TGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTA AA 22H5 N92QTCCTATGTGCTGACTCAGCCACCCTCGGTGT 423 light CAGTGGCCCCAGGACAGACGGCCAGGATTAchain CCTGTGGGGGAAACAACATTGGAAGTCAAA GTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTCCTGGTCGTCTATGATGATA GCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGTTCCAACTCTGGGAACACGGCCA CCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGG AT CAG ACTAGTGATCATGTGGTATTCGGCGGGGGGACCAAGCTGACCGTCCTAGGTCAGC CCAAGGCCAACCCCACTGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTCCAAGCCAACA AGGCCACACTAGTGTGTCTGATCAGTGACTTCTACCCGGGAGCTGTGACAGTGGCCTGGA AGGCAGATGGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCAAACCCTCCAAACAGAGC AACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCCGAGCAGTGGAAGTCCCAC AGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCC TACAGAATGTTCA 22H5 S94ATCCTATGTGCTGACTCAGCCACCCTCGGTGT 424 light CAGTGGCCCCAGGACAGACGGCCAGGATTAchain CCTGTGGGGGAAACAACATTGGAAGTCAAA GTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTCCTGGTCGTCTATGATGATA GCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGTTCCAACTCTGGGAACACGGCCA CCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGG ATAATACT GCT GATCATGTGGTATTCGGCGGGGGGACCAAGCTGACCGTCCTAGGTCAGC CCAAGGCCAACCCCACTGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTCCAAGCCAACA AGGCCACACTAGTGTGTCTGATCAGTGACTTCTACCCGGGAGCTGTGACAGTGGCCTGGA AGGCAGATGGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCAAACCCTCCAAACAGAGC AACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCCGAGCAGTGGAAGTCCCAC AGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCC TACAGAATGTTCA 22H5 C109SCAGGTCACCTTGAAGGAGTCTGGTCCTGTG 425 heavy CTGGTGAAACCCACAGAGACCCTCACGCTGchain ACCTGCACCGTGTCTGGGTTCTCACTCAGC AATGCTAGAATGGGTGTGAGCTGGATCCGTCAGCCCCCAGGGAAGGCCCTGGAGTGGCTT GCACACATTTTTTCGAATGACGAAAAATCCTACAGCACATCTCTGAAGAGCAGGCTCACC ATCTCCAAGGACACCTCCAAAAGCCAGGTGGTCCTTACCATGACCAACATGGACCCTGTG GACACAGCCACATATTACTGTGCACGGATATTATTAGTGGGAGCTTACTACTAC AGC GGT ATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCTAGTGCCTCCACCAAGGGCCCA TCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCT GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGA CCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCA GCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATC ACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGC CCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCC ACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCA AGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACC GTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGC CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACA GGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTG CCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC CGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTA CAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCG TGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTA AA 16H7 Insertion ofCAGGTCACCTTGAAGGAGTCTGGTCCTGTG 426 heavy Y107CTGGTGAAACCCACAGAGACCCTCACGCTG chain ACCTGCACCGTGTCTGGGTTCTCACTCAACAATGCTAGAATGGGTGTGAGCTGGATCCGT CAGCCCCCAGGGAAGGCCCTGGAGTGGCTTGCACACATTTTTTCGAATGACGAAAAATCC TACAGCACATCTCTGAAGAGCAGGCTCACCATCTCCAAGGACACCTCCAAAAGCCAGGTG GTCCTAATTATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACGGTCA GTAGTAACTGGCGGCTAC TATTACTACGACGGTATGGACGTCTGGGGCCAAGGGACCACG GTCACCGTCTCTAGTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCA GGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAAC CGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTG TCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTT CGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACA AGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAG GACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCC TGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTG GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCA ACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCA AGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATC TCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCG ACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCT CCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCA GGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACT ACACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAAA

TABLE 6C CDR Amino Acid Sequences of Variants Location CDR1 CDR2 CDR3Core of SEQ SEQ SEQ Sequence Variation Mutation CDR1 ID NO CDR2 ID NOCDR3 ID NO: 16H7 light Q16K FW GGNNIGSESV 166 DDSDRPS 176 QVWDGNSDHVV188 chain 16H7 light D49Y CDR2 GGNNIGSESVH 166 Y DSDRPS 427 QVWDGNSDHVV188 chain 16H7 light D49A CDR2 GGNNIGSESVH 166 A DSDRPS 428 QVWDGNSDHVV188 chain 16H7 light D91A CDR3 GGNNIGSESVH 166 DDSDRPS 176 QVW A GNSDHVV429 chain 16H7 light D49A + D91A CDR2, GGNNIGSESVH 166 A DSDRPS 427 QVWA GNSDHVV 430 chain CDR3 16H7 heavy V24F FW NARMGVS 122 HIFSNDEKSYSTSLKS133 SVVTGGYYYDGMDV 148 chain 16H7 heavy I83T FW NARMGVS 122HIFSNDEKSYSTSLKS 133 SVVTGGYYYDGMDV 148 chain 16H7 heavy V24F + I83T FWNARMGVS 122 HIFSNDEKSYSTSLKS 133 SVVTGGYYYDGMDV 148 chain 16H7 heavyE16Q + V24F + FW NARMGVS 122 HIFSNDEKSYSTSLKS 133 SVVTGGYYYDGMDV 148chain I83T 16H7 heavy E16Q + V24F + FW NARMGVS 122 HIFSNDEKSYSTSLKS 133SVVTGGYYYDGMDV 148 chain I83T + T19L 16H7 heavy E16Q + V24F + FW, CDR3NARMGVS 122 HIFSNDEKSYSTSLKS 133 I VVTGGYYYDGMDV 431 chain I83T +S100I + T119L 16H7 heavy I83K FW NARMGVS 122 HIFSNDEKSYSTSLKS 133SVVTGGYYYDGMDV 148 chain 16H7 heavy S100I CDR3 NARMGVS 122HIFSNDEKSYSTSLKS 133 I VVTGGYYYDGMDV 432 chain 16H7 heavy D88R + P89A +FW NARMGDV 122 HIFSNDEKSYSTSLKS 133 SVVTGGYYYDGMDV 148 chain V90E16H7 heavy D88R + P89A + FW, CDR3 NARMGVS 122 HIFSNDEKSYSTSLKS 133 IVVTGGYYYDGMDV 433 chain V90E + S101I 16H7 heavy Deletion CDR3 NARMGVS122 HIFSNDEKSYSTSLKS 133 SVVTGGYYDGMDV 434 chain of Y107 16H7 heavyD109S CDR3 NARMGVS 122 HIFSNDEKSYSTSLKS 133 SVVTGGYYY S GMDV 435 chain22H5 light N92Q CDR3 GGNNIGSQSVH 167 DDSDRPS 176 QVWD Q TSDHVV 436 chain22H5 light S94A CDR3 GGNNIGSQSVH 167 DDSDRPS 176 QVWDNT A DHVV 437 chain22H5 heavy C109S CDR3 NARMGVS 122 HIFSNDEKSYSTSLKS 133 ILLVGAYYY S GMDV438 chain 16H7 heavy Insertion CDR3 NARMGVS 122 HIFSNDEKSYSTSLKS 133SVVTGGYYYYDGMDV 439 chain of Y107

TABLE 6D CDR Nucleic Acid Sequences of Variants Location CDR1 CDR2 CDR3Core of SEQ SEQ SEQ Sequence Variation Mutation CDR1 ID NO CDR2 ID NOCDR3 ID NO: 16H7 light Q16K FW GGGGGAAACAACATT 239 GATGATAGCGACC 249CAGGTGTGGGATGG 260 chain GGAAGTGAAAGTGTG GGCCCTC TAATAGTGATCATG CACTGGTA 16H7 light D49Y CDR2 GGGGGAAACAACATT 239 TAT GATAGCGACC 442CAGGTGTGGGATGG 260 chain GGAAGTGAAAGTGTG GGCCCTCA TAATAGTGATCATG CACTGGTA 16H7 light D49A CDR2 GGGGGAAACAACATT 239 GCT GATAGCGACC 443CAGGTGTGGGATGG 260 chain GGAAGTGAAAGTGTG GGCCCTCA TAATAGTGATCATG CACTGGTA 16H7 light D91A CDR3 GGGGGAAACAACATT 239 GATGATAGCGACC 249CAGGTGTGG GCT GG 445 chain GGAAGTGAAAGTGTG GGCCCTCA TAATAGTGACCATG CACTGGTA 16H7 light D49A + D91A CDR2, GGGGGAAACAACATT 239 GCT GATAGCGACC444 CAGGTGTGG GCT GG 445 chain CDR3 GGAAGTGAAAGTGTG GGCCCTCATAATAGTGACCATG CAC TGGTA 16H7 heavy V24F FW AATGCTAGAATGGGT 196CACATTTTTTCGA 206 TCAGTAGTAACTGG 221 chain GTGAGC ATGACGAAAAATCCGGCTACTACTACG CTACAGCACATCTC ACGGTATGGACGTC TGAAGAGC 16H7 heavy I83T FWAATGCTAGAATGGGT 196 CACATTTTTTCGAA 206 TCAGTAGTAACTGG 221 chain GTGAGCTGACGAAAAATCCT CGGCTACTACTACG ACAGCACATCTCTG ACGGTATGGACGTC AAGAGC16H7 heavy V24F + I83T FW AATGCTAGAATGGGT 196 CACATTTTTTGACG 206TCAGTAGTAACTGG 221 chain GTGAGC AAAAATCCTACAGC CGGCTACTACTACGACATCTCTGAAGAG ACGGTATGGACGTC C 16H7 heavy E16Q + V24F + FWAATGCTAGAATGGGT 196 CACATTTTTTCGAA 206 TCAGTAGTAACTGG 221 chain I83TGTGAGC TGACGAAAAATCCT CGGCTACTACTACG ACAGCACATCTCTG ACGGTATGGACGTCAAGAGC 16H7 heavy E16Q + V24F + FW AATGCTAGAATGGGT 196 CACATTTTTTCGAA206 TCAGTAGTAACTGG 221 chain I83T + T19L GTGAGC TGACGAAAAATCCTCGGCTACTACTACG ACAGCACATCTCTG ACGGTATGGACGTC AAGAGC 16H7 heavy E16Q +V24F + FW, CDR3 AATGCTAGAATGGGT 196 CACATTTTTTCGAA 206 ATCGTAGTAACTGG446 chain I83T + S100I + GTGAGC TGACGAAAAATCCT CGGCTACTACTACG T119LACAGCACATCTCTG ACGGTATGGACGTC AAGAGC 16H7 heavy I83K FW AATGCTAGAATGGGT196 CACATTTTTTCGAA 206 TCAGTAGTAACTGG 221 chain GTGAGC TGACGAAAAATCCTCGGCTACTACTACG ACAGCACATCTCTG ACGGTATGGACGTC AAGAGC 16H7 heavy S100ICDR3 AATGCTAGAATGGGT 196 CACATTTTTTCGAA 206 ATCGTAGTAACTGG 446 chainGTGAGC TGACGAAAAATCCT CGGCTACTACTACG ACAGCACATCTCTG ACGGTATGGACGTCAAGAGC 16H7 heavy D88R + P89A + FW AATGCTAGAATGGGT 196 CACATTTTTTCGAA206 TCAGTAGTAACTGG 221 chain V90E GTGAGC TGACGAAAAATCCT CGGCTACTACTACGACAGCACATCTCTG ACGGTATGGACGTC AAGAGC 16H7 heavy D88R + P89A + FW, CDR3AATGCTAGAATGGGT 196 CACATTTTTTCGAA 206 ATCGTAGTAACTGG 446 chain V90E +S100I GTGAGC TGACGAAAAATCCT CGGCTACTACTACG ACAGCACATCTCTG ACGGTATGGACGTCAAGAGC 16H7 heavy Deletion CDR3 AATGCTAGAATGGGT 196 CACATTTTTTCGAA 206TCAGTAGTAACTGG 447 chain of Y107 GTGAGC TGACGAAAAATCCT CGGCTACTACGACGACAGCACATCTCTG GTATGGACGTC AAGAGC 16H7 heavy D109S CDR3 AATGCTAGAATGGGT196 CACATTTTTTCGAA 206 TCAGTAGTAACTGG 448 chain GTGAGC TGACGAAAAATCCTCGGCTACTACTACA ACAGCACATCTCTG GCGGTATGGACGTC AAGAGC 22H5 light N92Q CDR3GGGGGAAACAACATT 240 GATGATAGCGACCG 249 CAGGTGTGGGAT CA 449 chainGGAAGTCAAAGTGTG GCCCTCA G ACTAGTGATCATG CAC TGGTA 22H5 light S94A CDR3GGGGGAAACAACATT 240 GATGATAGCGACCG 249 CAGGTGTGGGATAA 450 chainGGAAGTCAAAGTGTG GCCCTCA TACT GCT GATCATG CAC TGGTA 22H5 heavy C109S CDR3AATGCTAGAATGGGT 196 CACATTTTTTCGAA 206 ATATTATTAGTGGG 451 chain GTGAGCTGACGAAAAATCCT AGCTTACTACTAC A ACAGCACATCTCTG GC GGTATGGACGTC AAGAGC16H7 heavy Insertion of CDR3 AATGCTAGAATGGGT 196 CACATTTTTTCGAA 206TCAGTAGTAACTGG 452 chain Y107 GTGAGC TGACGAAAAATCCT CGGCTAC TATTACTACAGCACATCTCTG ACGACGGTATGGAC AAGAGC GTC

Additionally, a “hemibody” was generated and studied. This structurecomprised the 16H7 light chain (L3; SEQ ID NO:50), which was paired withan engineered form of the 16H7 heavy chain; the engineered heavy chaincomprised the 16H7 heavy chain (SEQ ID NO:32) joined via a (G₄S)₈ linker(SEQ ID NO:440) to an IgG2 Fc sequence (SEQ ID NO:441), which pairedwith the Fc sequence of the 16H7 heavy chain. The component parts of thehemibody have the following sequences:

16H7 Heavy Chain (SEQ ID NO: 32)MDMRVPAQLLGLLLLWLRGARCQVTLKESGPVLVKPTETLTLTCTVSGFSLNNARMGVSWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKSQVVLIMTNMDPVDTATYYCARSVVTGGYYYDGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSP Linker(SEQ ID NO: 440) GGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS IgG2 Fc(SEQ ID NO: 441) ERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The full hemibody heavy chain had the amino acid sequence shown below:

(SEQ ID NO: 453) MDMRVPAQLLGLLLLSLRGARCQVTLKESGPVLVKPTETLTLTCTVSGFSLNNARMGVSWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKSQVVLIMTNMDPVDTATYYCARSVVTGGYYYDGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHANKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSLPGKwhich is encoded by the follow sequence:

(SEQ ID NO: 454) ATGGACATGAGGGTGCCCGCTCAGCTCCTGGGGCTCCTGCTGCTGTGGCTGAGAGGTGCGCGCTGTCAGGTCACCTTGAAGGAGTCTGGTCCTGTGCTGGTGAAACCCACAGAGACCCTCACGCTGACCTGCACCGTGTCTGGGTTCTCACTCAACAATGCTAGAATGGGTGTGAGCTGGATCCGTCAGCCCCCAGGGAAGGCCCTGGAGTGGCTTGCACACATTTTTTCGAATGACGAAAAATCCTACAGCACATCTCTGAAGAGCAGGCTCACCATCTCCAAGGACACCTCCAAAAGCCAGGTGGTCCTAATTATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACGGTCAGTAGTAACTGGCGGCTACTACTACGACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCTAGTGCCAGCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCCTGCAGCAGAAGCACCAGCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTCACCGTGCCCAGCAGCAACTTCGGCACCCAGACCTACACCTGTAACGTGGACCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTGGAGCGGAAGTCCAGCGTGGAGTGCCCTCCTTGTCCTGCCCCTCCTGTGGCCGGACCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCCGAGGTGCAGTTCAATTGGTACGTGGACGGGGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAACAGTTCAACAGCACCTTCCGGGTGGTGTCCGTCCTCACCGTGGTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTCTCCAACAAGGGCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGACCAAGGGCCAGCCTCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGGGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTCGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCATGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGTAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGAGGGGGCGGATCTGGTGGTGGAGGCAGCGGCGGAGGTGGAAGTGGCGGTGGAGGATCCGGTGGAGGCGGCTCAGGTGGCGGCGGAAGCGAGAGAAAGTCCTCCGTGGAGTGTCCACCATGCCCTGCTCCACCAGTGGCTGGCCCTTCCGTCTTTCTCTTTCCACCTAAACCTAAGGATACACTCATGATCTCCAGAACTCCAGAGGTCACATGTGTGGTCGTCGATGTCAGTCATGAGGATCCTGAAGTCCAGTTTAACTGGTATGTGGATGGCGTCGAAGTCCATAATGCTAAGACAAAACCTCGCGAAGAACAGTTTAACTCCACCTTTAGAGTCGTGAGCGTGCTGACAGTCGTCCATCAGGATTGGCTCAATGGGAAAGAATACAAATGTAAAGTCTCTAACAAAGGACTGCCCGCTCCTATCGAAAAGACCATCTCCAAAACAAAGGGGCAGCCCAGAGAGCCCCAGGTCTACACACTCCCACCCTCCAGAGAAGAGATGACAAAAAATCAGGTGTCACTCACCTGTCTGGTCAAGGGGTTTTACCCCTCCGACATTGCCGTGGAATGGGAATCCAATGGGCAGCCTGAAAACAATTATAAGACTACACCTCCTATGCTCGACTCTGATGGGAGTTTCTTTCTCTACTCTAAACTCACAGTGGATAAGTCTAGATGGCAGCAGGGGAATGTCTTTTCCTGCTCCGTCATGCATGAAGCTCTCCACAATCATTATACACAGAAGTCTTTGTCCCTGTCC CCCGGCAAG

Example 14.1 β-Klotho Binding ELISA for Engineered Antibodies

The engineered forms of 16H7 and 22H5 were tested for β-Klotho bindingusing an ELISA assay. Conditions for the ELISA were as follows.

Streptavidin coated Maxisorp plates were incubated with 2 μg/ml β-Klothoovernight at 4 degrees. Antibodies were added in 3-fold serial dilutionsstarting at 1 μM for 1 hour at room temp. HRP conjugated anti-human Fcwas used as the detector antibody. Signal was developed with Lumiglo andread on Envision.

Results of the ELISA assay are shown in FIG. 32A-32C and indicate thatmost variants of 16H7 bound to human β-Klotho except for a mutantcarrying insertion of tyrosine at position 107.

Example 14.2 Engineered Variants of 16H7 and 22H5Bind to Native ReceptorStructure, as Shown by FACS

A FACS binding assay was performed with several of the engineered formsof 16H7 and 22H5. The experiments were performed as follows.

CHO cells stably expressing FGF21 receptor were treated with parentantibody 16H7 and 22H5 and also with engineered variants of them (1 μgper 1×10⁶ cells in 100 μl PBS/0.5% BSA). Cells were incubated with theantibodies at 4° C. followed by two washes with PBS/BSA. Cells were thentreated with FITC-labeled secondary antibodies at 4° C. followed by twowashes. The cells were resuspended in 1 ml PBS/BSA and antibody bindingwas analyzed using a FACS Calibur instrument.

Consistent with ELISA results, most of engineered variants of FGF21receptor agonistic antibodies tested bind well to cell surface FGF21receptor in FACS. This observation further confirmed that the guidedengineering of FGF21 receptor agonistic antibodies maintain binding tothe native structure. In one mutant, in which CDR3 was engineered toinclude a tyrosine at position Y107, a complete loss of binding to cellsurface receptor was observed, which is similar to the ELISA results.This observation points to the role of CDR3 loop in binding to nativeconformation.

Example 14.3 Activity of 16H7 and 22H5 Variants in Primary HumanAdipocytes

FGF21 stimulates glucose uptake and lipolysis in cultured adipocytesand, therefore, adipocytes are often considered to be a physiologicallyrelevant assay. A panel of the engineered variants of 16H7 and 22H5 wasshown to exhibit Erk-phosphorylation activity similar to FGF21 in thehuman adipocyte assay with an estimated EC50 less than 10 nM, as shownin Table 7.

TABLE 7 Activity of Variants in Human Adipocyte Assay SEQ ID NO of EC50Core Sequence Variant Chain Variant (nM) 16H7 Heavy Chain 391 I83T 0.7316H7 Heavy Chain 393 E16Q + V24F + I83T 0.38 16H7 Heavy Chain 398 D88R +P89A + V90E 0.35 16H7 Heavy Chain 394 E16Q + V24F + I83T + 0.36 T119L16H7 (WT) 0.53 22H5 Light Chain 403 S94A 1.98 22H5 Light Chain 402 N92Q3.33 16H7 Heavy Chain 400 Deletion of Y107 1.04 16H7 Heavy Chain 396I83K 0.39 16H7 Heavy Chain 397 S100I 0.17 16H7 Heavy Chain 401 D109S0.31 16H7 Heavy Chain 399 D88R + P89A + V90E + 0.14 S100I 16H7 HeavyChain 395 E16Q + V24F + I83T + 0.24 S100I + T119L 22H5 Heavy Chain 405Insertion of Y107 0.51 16H7 Heavy Chain 390 V24F 0.75 16H7 Heavy Chain392 V24F + I83T 0.37 16H7 Light Chain 386 D49Y 0.60 16H7 Light Chain 387D49A 0.63 16H7 Light Chain 389 D49A, D91A 1.4 16H7 Light Chain 388 D91A1.3 16H7 Light Chain 385 Q16K 0.11 22H5 (WT) 2.27

Example 14.4 Biacore™ Binding Experiments and Off-Rate Measurement

Binding of 16H7 and 22H5 variants to human β-Klotho was tested usingBiacore™ assays. Briefly, mouse anti-His antibody (Qiagen™, Valencia,Calif.) was immobilized on a CM5 chip using amine coupling reagents(General Electronics™, Piscataway, N.J.). His-tagged humanrecombinantβ-Klotho was captured on the second flow cell to ˜100 RU. Thefirst flow cell was used as a background control. 100 nM mAbs werediluted in PBS plus 0.1 mg/ml BSA, 0.005% P20 and injected over theβ-Klotho captured on anti-His antibody surface. For kinetic measurement,0.78˜100 nM mAbs diluted in PBS plus 0.1 mg/ml BSA, 0.005% P20 wereinjected over the β-Klotho surface.

The variants tested are summarized in Table 8:

TABLE 8 Variants Studied in Binding and Off-rate Experiments Core HeavyLight Antigen Chain Chain Construct Binding Heavy Chain Light Chain SEQID SEQ ID Number Protein Identifier/Variation Identifier/Variation NO NO22H5 H2 L2 31 13  #1, P60881.3 16H7 I83T L3 391 14  #2, P60880.3 16H7E16Q + V24F + I83T L3 393 14  #3, P60890.3 16H7 D88R + P89A + V90E L3398 14  #4, P60878.3 16H7 E16Q + V24F + I83T + L3 394 14 T119L  #5, 16H7WT 16H7 H3 L3 32 14  #6, P60898.3 22H5 H2 S94A 31 403  #7, P60897.3 22H5H2 N92Q 31 402  #8, P60886.3 16H7 Deletion of Y107 L3 400 14  #9,P60885.3 16H7 I83K L3 396 14 #10, P60884.3 16H7 S100I L3 397 14 #11,P60883.3 16H7 D109S L3 401 14 #12, P60879.3 16H7 D88R + P89A + V90E + L3399 14 S100I #13, P60882.3 16H7 Hemibody Heavy L3 453 14 Chain #14,P60891.3 16H7 E16Q + V24F + I83T + L3 395 14 S100I + T119L #15, P60889.316H7 Insertion of Y107 L3 405 14 #16, P60888.3 16H7 V24F L3 390 14 #17,P60887.3 16H7 V24F + I83T L3 392 14 #18, P60894.3 16H7 H3 D49Y 32 386#19, P60895.3 16H7 H3 D49A 32 387 #20, P60893.3 16H7 H3 D49A + D91A 32389 #21, P60892.3 16H7 H3 D91A 32 388 #22, P60896.3 16H7 H3 Q16K 32 385#23, P60899.2 22H5 C109S L2 404 13

Among the engineered mAbs tested, the majority of them showed tightbinding to human β-Klotho, except #15 which showed no binding. Table 9below shows 100 nM mAbs binding to β-Klotho captured on anti-His. FIG.33 shows the comparison to off-rate.

TABLE 9 Binding to β-Klotho Sample koff (1/s) #20, P60893.3 1.9E−04 #11,P60883.3 2.6E−04 #23, P60899.2 3.0E−04 22H5 3.1E−04 #18, P60894.33.1E−04  #6, P60898.3 3.5E−04 #13, P60882.3 4.4E−04  #7, P60897.35.2E−04  #8, P60886.3 5.3E−04

Example 15 Combinations of Antigen Binding Proteins Show an AdditiveEffect

Antigen binding proteins representing different binding bins (FIG. 11 aand b) were selected and tested in reporter assays in pairs to determineif the pair of molecules would behave in an additive fashion. Assayswere run as follows.

On day one, AM-1/D FGFR1c+β-Klotho Luc clone was seeded in a 96-wellplate at 20K cells/well in DMEM+10% FBS medium. The plate was incubatedovernight. The following day, the medium was replaced with assay medium(DMEM+0.2% FBS) and incubated overnight. From an antibody working stock(1 mg/mL in PBS), each antibody under study was prepared at a dilutionof 2 μg/ml in assay medium. 100 μL of each antibody to be tested wascombined in a U-bottom plate. The assay medium was removed from thecells, and 50 μL of the antibody mixtures was transferred to the cells.The antibody mixtures were incubated on the cells for 5 hrs. Lastly,each sample was read-out with SteadyGlo Luciferase reagent (50 μl/well),per the manufacturer's specifications.

Table 10 below is a summary of the activity (% of FGF21 activity fromthe reporter assay) observed from the study; Table 11 expresses theobserved activities with respect to bins.

TABLE 10 Antibody Combination Activity (%) Iso 6 5 4 3 2 1 IgG2k 2G1016H7 12E4.1 20D4.1 39F7 26H11.1 Iso IgG2k ND −1.1 23.5 25.4 12.5 9.217.9 1 26H11.1 17.9 19.1 36.7 21.4 28.3 20.7 2 39F7 9.2 9.1 37.0 30.821.4 3 20D4.1 12.5 13.5 19.4 32.0 4 12E4.1 25.4 28.8 41.5 5 16H7 23.527.8 6 2G10 −1.1

TABLE 11 Antibody Combinations Expressed in Terms of Bins D C B B A ABin Ab ID Isotype 2G10 39F7 12E4.1 26H11.1 16H7 20D4.1 A 20D4.1 12.513.5 21.4 32.0 28.3 19.4 A 16H7 23.5 27.8 37.0 41.5 36.7 B 26H11.1 17.919.1 20.7 21.4 B 12E4.1 25.4 28.8 30.8 C 39F7 9.2 9.1 D 2G10 −1.1

Surprisingly, several pairs of molecules showed an additive effect. Asshown in FIGS. 34 and 35, respectively, 39F11 and FGF21 showed anadditive effect when measured in the reporter assay of Example 5, as did16H7 and 39H11.

Summarizing the data from this set of experiments, it was observed thatantigen binding proteins from the same binding bin, e.g., 16H7 whenpaired with 20D4 (both from Group A), the summed activity was notadditive. This was also observed when 12E4 was paired with 26H11 (bothfrom Group B). Additionally, paired antigen binding proteins fromnon-overlapping bins showed additive activities, e.g., 16H7 (Group A)paired with 26H11 or 12E4 (Group B), or paired with 39F7 (Group C).Further, antigen binding proteins 26H11 and 12E4 (Group B) showedadditive effect when combined with Abs from Group A but not Group C,suggesting there may be some overlap between the binding sites of GroupB and Group C and/or that the activation conformations induced by theantigen binding proteins from Group B and Group C are not mutuallycompatible. Finally, as expected, when a functional antigen bindingprotein is paired with a non-functional antigen binding protein (e.g.,2G10) which binds to a distinct and non-overlapping binding site fromGroup A, B or C, there is no effect upon the activity from thefunctional antigen binding protein from Group A, B or C.

Collectively, this data suggests that the disclosed antigen bindingproteins can be co-administered to enhance the effect that a givenantigen binding protein may provide on its own.

Each reference cited herein is incorporated by reference in its entiretyfor all that it teaches and for all purposes.

The present disclosure is not to be limited in scope by the specificembodiments described herein, which are intended as illustrations ofindividual aspects of the disclosure, and functionally equivalentmethods and components form aspects of the disclosure. Indeed, variousmodifications of the disclosure, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

What is claimed is:
 1. An isolated monoclohal antibody that specificallybinds to human B-Klotho polypeptide comprising SEQ ID NO: 7, whereinsaid antibody comprises heavy chain variable region comprisingcomplementarity determining regions (CDRs) comprising the amino acidsequences of SEQ ID NO: 122, SEQ ID NO: 133, and SEQ ID NO: 148 and alight chain variable region comprising CDRs comprising the amino acidsequences of SEQ ID NO: 166, SEQ ID NO: 176, SEQ ID NO:
 188. 2. Theantigen binding protein of claim 1, wherein the antigen binding proteinis a human antibody, a humanized antibody, chimeric antibody, amonoclonal antibody, a polyclonal antibody, a recombinant antibody, anantigen-binding antibody fragment, a single chain antibody, a diabody, atriabody, a tetrabody, a Fab fragment, an F(fab′)₂ fragment, a domainantibody, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, or anIgG4 antibody having at least one mutation in the hinge region.
 3. Apharmaceutical composition comprising the isolated monoclonal antibodyof claim 1 and a pharmaceutically acceptable carrier.